Perlecan as a biomarker for renal dysfunction

ABSTRACT

The application discloses PERLECAN as a new biomarker for renal dysfunction; methods for predicting, diagnosing, prognosticating and/or monitoring said dysfunction based on measuring said biomarker; and kits and devices for measuring said biomarker and/or performing said methods.

FIELD OF THE INVENTION

The invention relates to protein- and/or peptide-based biomarkers usefulfor predicting, diagnosing, prognosticating and/or monitoring diseasesand conditions in subjects, in particular renal dysfunction; and torelated methods, kits and devices.

BACKGROUND OF THE INVENTION

In many diseases and conditions, a favourable outcome of prophylacticand/or therapeutic treatments is strongly correlated with early and/oraccurate prediction, diagnosis, prognosis and/or monitoring of a diseaseor condition. Therefore, there exists a continuous need for additionaland preferably improved manners for early and/or accurate prediction,diagnosis, prognosis and/or monitoring of diseases and conditions toguide the treatment choices.

The mammalian renal system plays central roles inter alia in the removalof catabolic waste products from the bloodstream and in the maintenanceof fluid and electrolyte balances in the body.

Renal dysfunction encompasses diseases and conditions in which kidneyfunction is inadequate, such as for example diseases and conditionscharacterised by an acute or chronic deterioration of kidney function,more particularly characterised by an acute or chronic decline in kidneyexcretory function, as evidenced for example by reduced glomerularfiltration rate. Renal dysfunction may develop into a life-threateningcondition in which the (systemic) build-up of catabolic waste productsand other harmful or toxic substances and/or the development ofsignificant imbalances in bodily fluids or electrolytes may lead to,contribute to or exacerbate the failure of other major organ systems anddeath.

Signs and symptoms of renal dysfunction may include inter alia increasedlevels of urea in the blood, volume overload and swelling, abnormal acidlevels, abnormal levels of potassium, calcium and/or phosphate, changesin urination, fatigue, skin rash or itching, nausea, dyspnea, reducedkidney size, haematuria and anaemia. However, renal dysfunction isfrequently insidious and may progress to an advanced stage before thepatient notices problems and decides to seek a physician. Therefore,renal dysfunction is commonly diagnosed late, and the patient mayalready be in need of radical and non-trivial treatments such asdialysis or kidney transplantation.

To aid diagnosis of renal dysfunction, some methods have been developedpreviously. For example, one way is to determine the glomerularfiltration rate (GFR). However, GFR measurements rely on invasive,time-consuming and expensive procedures involving the injection ofexogenous and potentially harmful diagnostic substances and measuringtheir excretion at specified time period(s). Another method is tomeasure serum creatinine clearance. Creatinine originates from muscletissue and is increasingly secreted by renal tubules concomitant withdecreasing renal function. However, serum creatinine levels depend onage, sex, diet, muscle mass, ethnic background, physical activity,disease, other manners of secretion, etc., which factors may impair thereliability of creatinine clearance for diagnosis of renal dysfunction.A further endogenous biomarker for diagnosing renal dysfunction isCystatin C. Advantageously, compared to creatinine the expression ofCystatin C is comparably steady. Nevertheless, Cystatin C does show somelimitations: for example, its levels are affected by immunosuppressivetherapeutics and are dependent on thyroid function. Cystatin C also doesnot react rapidly enough to acute changes in GFR and is thus not asatisfactory marker for acute kidney injury (AKI). Another endogenousmarker is neutrophil gelatinase-associated lipocalin (NGAL) whichappears to detect early stages of acute renal injury. However, the useof NGAL is confounded by its anti-inflammatory role, which may lead tosubstandard specificity in complicated patient populations.

Furthermore, the timely (i.e. early) discovery of the need of renalreplacement therapy (RRT) in postoperative or critically ill patientsprone to develop kidney related problems is fundamental to reducemortality. To date however there is no objective early measure for theneed of RRT initiation and a biomarker that can specifically identifythose patients that will benefit from early RRT is highly anticipated.

Dependable and preferably early detection and intervention is criticalto effective treatment of renal dysfunction. Consequently, provision offurther, alternative and preferably improved markers and tools fordiagnosis, prediction, prognosis and/or monitoring of renal dysfunctioncontinues to be of prime importance.

The present invention addresses the above needs in the art byidentifying biomarkers for renal dysfunction and related diseases andconditions and providing uses therefore.

SUMMARY OF THE INVENTION

Having conducted extensive experiments and tests, the inventors havefound that levels of the protein PERLECAN, more precisely theEndorepellin part thereof and even more precisely the LG-3 domainthereof (called generally “perlecan” hereinafter) in blood samples areclosely indicative of kidney function. In particular, in clinicalsamples from 299 patients PERLECAN showed a significant association withseveral tested clinical parameters related to kidney function, amongothers estimated glomerular filtration rate (eGFR), creatinine levels,blood urea nitrogen (BUN) levels, history of kidney failure and CystatinC levels.

Further, for discriminating subjects with decreased GFR (<60 ml/min/1.73m²) from subjects with normal GFR, the median AUC value (area under theROC curve; “ROC” stands for receiver operating characteristic) is atleast comparable between PERLECAN (0.91) and Cystatin C (0.92). The AUCvalue is a combined measure of sensitivity and specificity and a higherAUC value (i.e., approaching 1) in general indicates an improvedperformance of the test.

As mentioned, throughout the specification the term “PERLECAN” mayencompass PERLECAN and fragments of PERLECAN, such as the Endorepellinor LG3 domain of PERLECAN. Hence, reference to “PERLECAN” as used hereinmay also be read as “PERLECAN or a fragment thereof”. Preferably, saidfragment may be the Endorepellin domain of PERLECAN. Further preferably,said fragment may be the LG3 domain of PERLECAN.

Accordingly, the inventors have realised PERLECAN as a new biomarkeradvantageous for evaluating renal function.

Further provided is a method for determining renal function in a subjectcomprising measuring the quantity of PERLECAN in a sample from saidsubject. Particularly provided is a method for predicting, diagnosing,prognosticating and/or monitoring renal dysfunction in a subjectcomprising measuring PERLECAN levels in a sample from said subject. Asused throughout this specification, measuring the levels of PERLECANand/or other biomarker(s) in a sample from a subject may particularlydenote that the examination phase of a method comprises measuring thequantity of PERLECAN and/or other biomarker(s) in the sample from thesubject. One understands that methods of prediction, diagnosis,prognosis and/or monitoring of diseases and conditions generallycomprise an examination phase in which data is collected from and/orabout the subject.

In an embodiment, a method for predicting, diagnosing and/orprognosticating renal dysfunction comprises the steps of: (i) measuringthe quantity of PERLECAN in a sample from the subject; (ii) comparingthe quantity of PERLECAN measured in (i) with a reference value of thequantity of PERLECAN, said reference value representing a knownprediction, diagnosis and/or prognosis of renal dysfunction or normalrenal function; (iii) finding a deviation or no deviation of thequantity of PERLECAN measured in (i) from the reference value; and (iv)attributing said finding of deviation or no deviation to a particularprediction, diagnosis and/or prognosis of renal dysfunction or normalrenal function in the subject.

The method for predicting, diagnosing and/or prognosticating renaldysfunction, and in particular such method comprising steps (i) to (iv)as set forth in the previous paragraph, may be performed for a subjectat two or more successive time points and the respective outcomes atsaid successive time points may be compared, whereby the presence orabsence of a change between the prediction, diagnosis and/or prognosisof renal dysfunction at said successive time points is determined. Themethod thus allows to monitor a change in the prediction, diagnosisand/or prognosis of renal dysfunction in a subject over time.

In an embodiment, a method for monitoring renal dysfunction comprisesthe steps of: (i) measuring the quantity of PERLECAN in samples from asubject from two or more successive time points; (ii) comparing thequantity of PERLECAN between the samples as measured in (i); (iii)finding a deviation or no deviation of the quantity of PERLECAN betweenthe samples as compared in (ii); and (iv) attributing said finding ofdeviation or no deviation to a change in renal function or renaldysfunction in the subject between the two or more successive timepoints. The method thus allows to monitor renal dysfunction or renalfunction in a subject over time. This monitoring can be important todetermine the start, type and/or continuation or change in treatment ofthe renal dysfunction.

Throughout the present disclosure, methods suitable for monitoring anyone condition or disease as taught herein can inter alia allow topredict the occurrence of the condition or disease, or to monitor theprogression, aggravation, alleviation or recurrence of the condition ordisease, or response to treatment or to other external or internalfactors, situations or stressors, etc. Advantageously, monitoringmethods as taught herein may be applied in the course of a medicaltreatment of the subject, preferably medical treatment aimed atalleviating the so-monitored condition or disease. Such monitoring maybe comprised, e.g., in decision making whether a patient may bedischarged, needs a change in treatment or needs further hospitalisationor requires Renal Replacement Therapy (RRT) to be initiated.

Similarly, throughout the present disclosure, methods suitable forprognosticating any one condition or disease as taught herein can interalia allow to prognosticate the occurrence of the condition or disease,or to prognosticate the progression, aggravation, alleviation orrecurrence of the condition or disease, or response to treatment or toother external or internal factors, situations or stressors, etc. mayallow to prognosticate

As shown in the experimental section, clinical parameters typifyingkidney dysfunction, such as for example reduced eGFR and elevatedCystatin C levels, associate with elevated levels of PERLECAN.Consequently, prediction or diagnosis of renal dysfunction or a poorprognosis of renal dysfunction can in particular be associated with anelevated level of PERLECAN.

For example but without limitation, an elevated quantity (i.e., adeviation) of PERLECAN in a sample from a subject compared to areference value representing the prediction or diagnosis of no renaldysfunction (i.e., normal renal function) or representing a goodprognosis for renal dysfunction respectively indicates that the subjecthas or is at risk of having renal dysfunction or indicates a poorprognosis for renal dysfunction in the subject (such as, e.g., aprognosis that a chronic renal dysfunction patient will progress towardsend-stage kidney disease).

Renal dysfunction may be characterised by reduced GFR or eGFR.(Estimated) glomerular filtration rate may be said to be reducedcompared to normal, if the GFR or eGFR is below normal by any extent.For example but without limitation: normal GFR or eGFR indicative ofnormal kidney function may denote values greater than 90 ml/min/1.73 m²;intermediate GFR or eGFR indicative of slightly impaired kidney functionmay denote values between 60 and 90 ml/min/1.73 m²; and reduced GFR oreGFR indicative of seriously impaired kidney function may denote valueslower than 60 ml/min/1.73 m².

In an exemplary but non-limiting experiment PERLECAN levels providedsatisfactory discrimination between normal and reduced GFR when thethreshold between normal and reduced GFR was set at 60 ml/min/1.73 m².Hence, in embodiments a threshold for normal vs. reduced GFR or eGFR maybe set at a value between about 50 and about 70 ml/min/1.73 m², e.g.,between about 55 and about 65 ml/min/1.73 m², e.g., at 55, 56, 57, 58,59, 60, 61, 62, 63, 64 or 65 ml/min/1.73 m², and preferably at 60ml/min/1.73 m² wherein a value above said threshold reflects normal GFRor eGFR and a value below said threshold denotes reduced GFR or eGFR.

In other embodiments a threshold for normal vs. reduced GFR or eGFR maybe set at a value between about 80 and about 100 ml/min/1.73 m², e.g.,between about 85 and about 95 ml/min/1.73 m², e.g., at 85, 86, 87, 88,89, 90, 91, 92, 93, 94 or 95 ml/min/1.73 m², and preferably at 90ml/min/1.73 m² wherein a value above said threshold reflects normal GFRor eGFR and a value below said threshold denotes reduced GFR or eGFR.

In an exemplary but non-limiting experiment PERLECAN levels providedsatisfactory discrimination between normal, intermediate and reduced GFRwhen the threshold between normal and intermediate GFR was set at 90ml/min/1.73 m² and the threshold between intermediate and reduced GFRwas set at 60 ml/min/1.73 m²

Hence, in yet other embodiments, a threshold for intermediate vs.reduced GFR or eGFR may be set at a value between about 50 and about 70ml/min/1.73 m², e.g., between about 55 and about 65 ml/min/1.73 m²,e.g., at 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 ml/min/1.73 m²,and preferably at 60 ml/min/1.73 m² wherein a value above said thresholdreflects intermediate GFR or eGFR and a value below said thresholddenotes reduced GFR or eGFR; and a further threshold for normal vs.intermediate GFR or eGFR may be set at a value between about 80 andabout 100 ml/min/1.73 m², e.g., between about 85 and about 95ml/min/1.73 m², e.g., at 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95ml/min/1.73 m², and preferably at 90 ml/min/1.73 m² wherein a valueabove said threshold reflects normal GFR or eGFR and a value below saidthreshold denotes intermediate GFR or eGFR.

As taught herein, the level of PERLECAN, such as for example thePERLECAN concentration in plasma and/or urine, correlates withglomerular filtration rate (GFR). Consequently, the quantity of PERLECANas measured in a subject can be converted to a GFR value in order todetermine or estimate the latter. A suitable conversion formula for suchpurpose may also include additional factors such as clinical parameters(without limitation, height, age, sex, race, muscle mass, etc.) and/orclinical variables (e.g., blood-measured variables such as withoutlimitation hematocrite, albumin concentration, thyroid hormones, etc.).

Consequently, an aspect provides a method for determining glomerularfiltration rate (GFR) of a subject comprising measuring the quantity ofPERLECAN in a sample from said subject and converting said measuredquantity of PERLECAN to GFR of said subject.

The quantity of PERLECAN as measured in a subject may be converted to aGFR value as a part or step of the herein disclosed diagnosis,prediction, prognosis and/or monitoring methods. So-calculated GFRvalues may be compared with known GFR values representing various stagesof GFR and kidney function impairment. The quantity of PERLECAN may thusbe used to determine the degree of GFR reduction in a subject.

Accordingly, in an embodiment of the herein disclosed diagnosis,prediction, prognosis and/or monitoring methods the renal dysfunctionmay encompass, denote or correspond to GFR reduction.

Also disclosed is a method to determine whether a subject is or will beor is not or will not be (such as, for example, still is, or is nolonger) in need of a therapy to treat renal dysfunction, comprising: (i)measuring the quantity of PERLECAN in the sample from the subject; (ii)comparing the quantity of PERLECAN measured in (i) with a referencevalue of the quantity of PERLECAN, said reference value representing aknown diagnosis, prediction and/or prognosis of renal dysfunction ornormal renal function; (iii) finding a deviation or no deviation of thequantity of PERLECAN measured in (i) from said reference value; (iv)inferring from said finding the presence or absence of a need for atherapy to treat renal dysfunction. A therapy may be particularlyindicated where steps (i) to (iii) allow for a conclusion that thesubject has or is at risk of having renal dysfunction or has a poorprognosis for renal dysfunction, such as for example but withoutlimitation, where the quantity of PERLECAN in the sample from thesubject is elevated (i.e., a deviation) compared to a reference valuerepresenting the prediction or diagnosis of no renal dysfunction (i.e.,normal renal function). Without limitation, a patient having renaldysfunction upon admission to or during stay in a medical care centremay be tested as taught herein for the necessity of continuing atreatment of said renal dysfunction, and may be discharged when suchtreatment is no longer needed or is needed only to a given limitedextent.

Particularly, the method may allow to determine whether a subject is orwill be or is not or will not be in need of early initiation of saidtherapy. For example, such determination using PERLECAN may be madeearlier and/or may allow for earlier initiation of the therapy comparedto currently available markers such as creatinine and urea (blood ureanitrogen).

Exemplary therapies for renal dysfunction encompass without limitationlow-potassium and/or low phosphorus diets, phosphorus-loweringmedications (e.g., calcium carbonate, calcitriol, sevelamer), red bloodcell production stimulating agents (e.g., erythropoietin, darbepoietin),iron supplements, blood pressure medications, vitamin supplements,haemodialysis, ultrafiltration, peritoneal dialysis, and kidneytransplantation. The need for RRT (such as haemodialysis,ultrafiltration or peritoneal dialysis) is typically important to testin postoperative or critically ill patients. PERLECAN is shown herein tobe a good early marker of the need for RRT and can hence help todecrease mortality in said patients by timely taking over the fluidfiltration function of the kidneys.

Hence, in an embodiment, the therapy to treat renal dysfunction may behaemodialysis, ultrafiltration or peritoneal dialysis in postoperativeand/or critically ill patients.

In an embodiment, the timing or scheduling of dialysis in a chronicdialysis patient may be informed by alterations in the quantity ofPERLECAN measured in said patient over time. For example, dialysis maybe scheduled if a predetermined change in PERLECAN quantity is detectedin such patient over time.

In embodiments, renal dysfunction as used herein may refer to acuterenal failure (acute kidney injury). In other embodiments, renaldysfunction as used herein may refer to chronic renal failure (chronickidney disease). In further embodiments, renal dysfunction as usedherein may be associated or caused by fibrosis of the kidney tissue(renal fibrosis), particularly but without limitation in chronic kidneydisease patients or heart failure patients.

Particularly advantageously, renal dysfunction as intended herein mayinvolve acute renal dysfunction or AKI. As demonstrated by theinventors, PERLECAN can detect abrupt changes in renal function. SinceAKI commonly entails sudden drops in GFR, the measurement of PERLECAN—asa marker rapidly reacting to such abrupt GFR changes—may be particularlysuitable for diagnosing, predicting, prognosticating and/or monitoringAKI.

Using PERLECAN as a marker for AKI may be particularly useful inpatients known or expected to be at risk of developing AKI. Withoutlimitation, such PERLECAN testing or screening may be effected in thegeneral population of intensive care unit (ICU) patients (i.e., testinga subject at ICU), such as, e.g., in patients having undergone surgeryand more particularly cardiac surgery, in whom the incidence of acutekidney injury can be as high as 30-50%. Also without limitation,PERLECAN testing or screening may be employed in patients undergoing orhaving undergone coronary or peripheral angiography, in whom theincidence of developing contrast fluid-induced nephropathy may be ashigh as 5-10%. By means of example, in such situations PERLECAN may beused as a diagnostic marker (e.g., PERLECAN may be measured within agiven time, e.g., within 24 hours, following the procedure) or as apredictive marker to identify patients sensitive or prone to AKIdevelopment.

As demonstrated in the examples, PERLECAN can identify subjects havingrenal dysfunction in a subject population presenting with (acute)dyspnea. Dyspnea (dyspnoea or shortness of breath) is a common anddistressing symptom which may be connected to a range of underlyingpathologies, such as, e.g., lung cancer, chronic obstructive pulmonarydisease (COPD), congestive or acute heart failure, and renaldysfunction. To treat a patient manifesting with dyspnea adequately, theunderlying problem needs to be established.

Accordingly, in methods of diagnosing, predicting, prognosticatingand/or monitoring renal dysfunction as taught herein, the subject maypresent with (manifest with) dyspnea. Preferably, the dyspnea may beacute dyspnea. Said methods may particularly allow to discriminatebetween (subjects having) dyspnea associated with or caused by renaldysfunction and (subjects having) dyspnea associated with or caused byother conditions (such as without limitation COPD or pneumonia).

As stated in the examples, the correlations between PERLECAN levels andCystatin C levels or eGFR persist even following a correction for thepresence of acute decompensated heart failure (AHF) in the subjectpopulation. Hence, PERLECAN can detect abrupt changes in renal function(eGFR) due to acute decompensation of the heart (i.e., reduced cardiacoutput).

Accordingly, in methods of diagnosing, predicting, prognosticatingand/or monitoring renal dysfunction as taught herein, the subject mayhave or may be at risk of having heart failure, preferably acutedecompensated heart failure (AHF). Such methods may inter alia allow todiagnose acute worsening of renal function associated with or caused byreduced cardiac output, or monitor renal function in the course oftreatment of AHF.

As also shown in the examples, the inventors have found that PERLECANlevels upon admission in subjects manifesting with acute dyspnea weresignificantly higher in those subjects who will have died within oneyear post-admission compared to those subjects who will have remainedalive at one year. This distinction was also observed when the patientpopulation was divided based on the presence or absence of acute heartfailure (AHF), or based on renal (dys)function as measured by GFR.Consequently, the inventors have realised PERLECAN as a new biomarkeradvantageous for predicting or prognosticating mortality in patientswith dyspnea, particularly acute dyspnea, in patients with AHF and/or inpatients with renal dysfunction, particularly chronic renal dysfunction.

Hence, provided is also a method for predicting or prognosticatingmortality in a subject having dyspnea and/or acute heart failure and/orrenal dysfunction, comprising measuring the quantity of PERLECAN in asample from said subject. Preferably, the dyspnea may be acute dyspnea.Preferably, the renal dysfunction may be chronic renal dysfunction,particularly chronic kidney disease. Without limitation, the dyspnea maybe associated with or caused by AHF and/or by renal dysfunction; or thedyspnea may be associated with our caused by conditions other than AHFand renal dysfunction; or the subject may have AHF and/or renaldysfunction without dyspnea symptoms.

In an embodiment, the method for predicting or prognosticating mortalityin a subject having dyspnea and/or acute heart failure and/or renaldysfunction comprises the steps of: (i) measuring the quantity ofPERLECAN in a sample from the subject; (ii) comparing the quantity ofPERLECAN measured in (i) with a reference value of the quantity ofPERLECAN, said reference value representing a known prediction orprognosis of mortality; (iii) finding a deviation or no deviation of thequantity of PERLECAN measured in (i) from the reference value; and (iv)attributing said finding of deviation or no deviation to a particularprediction or prognosis of mortality in the subject.

The present methods for predicting or prognosticating mortality may bepreferably performed for a subject once the subject presents with or isdiagnosed with dyspnea and/or acute heart failure and/or renaldysfunction, more preferably upon the initial (first) presentation ordiagnosis of said diseases and conditions.

As shown in the experimental section, increased mortality rate inpopulations of dyspneic and/or AHF and/or renal failure subjects isassociated with elevated levels of PERLECAN. Consequently, prediction orprognostication of increased mortality (increased risk or chance ofdeath within a predetermined time interval) can in particular beassociated with an elevated level of PERLECAN.

For example but without limitation, an elevated quantity (i.e., adeviation) of PERLECAN in a sample from a subject compared to areference value representing the prediction prognosis of a givenmortality (i.e., a given, such as a normal, risk or chance of deathwithin a predetermined time interval) indicates that the subject has acomparably greater risk of deceasing within said time interval.

Without limitation, mortality may be suitably expressed as the chance ofa subject to decease within an interval of for example several months orseveral years from the time of performing a prediction orprognostication method, e.g., within about 6 months or within about 1year or within about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9 or about 10 years from the time of performing theprediction or prognostication method.

In an exemplary but non-limiting experiment PERLECAN levels providedsatisfactory discrimination between normal and increased mortality indyspnea, in AHF, and in renal dysfunction subjects when the timeinterval for considering the alive vs. dead status was set at 1 yearfrom the time of performing the prediction or prognostication method.Hence, in embodiments mortality may be suitably expressed as the chanceof a subject to decease within an interval of between 6 months and 2years and preferably within 1 year from performing the prediction orprognostication method.

It shall be appreciated that finding of increased chance of death in asubject can guide therapeutic decisions to treat the subject's diseasesor conditions.

The inventors have further found that levels of PERLECAN protein areincreased in hypertrophied left ventricles of thoracic aorticconstriction (TAC) animals compared to controls. Accordingly, theinventors have realised PERLECAN as a new biomarker advantageous forevaluating left ventricular hypertrophy and cardiac fibrosis. WO2008/046509 studies the expression of PERLECAN on mRNA level in the DOCArat model of left ventricular hypertrophy, without a conclusive result.

Another aspect provides PERLECAN as a new biomarker advantageous forevaluating preeclampsia (PE). A further aspect provides PERLECAN as anew biomarker advantageous for evaluating proteinuria, e.g. associatedwith pregnancy (PAP), or associated with Metabolic syndrome or Type IIdiabetes.

Hence, provided are methods for predicting, diagnosing, prognosticatingand/or monitoring any one of left ventricular hypertrophy (LVH), cardiacfibrosis (CF), PE or PAP in a subject comprising measuring PERLECANlevels in a sample from said subject.

In an embodiment, a method for predicting, diagnosing and/orprognosticating any one of LVH, CF, PE or PAP comprises the steps of:(i) measuring the quantity of PERLECAN in a sample from the subject;(ii) comparing the quantity of PERLECAN measured in (i) with a referencevalue of the quantity of PERLECAN, said reference value representing aknown prediction, diagnosis and/or prognosis of LVH, CF, PE or PAP;(iii) finding a deviation or no deviation of the quantity of PERLECANmeasured in (i) from the reference value; and (iv) attributing saidfinding of deviation or no deviation to a particular prediction,diagnosis and/or prognosis of LVH, CF, PE or PAP in the subject.

The method for predicting, diagnosing and/or prognosticating any one ofLVH, CF, PE or PAP, and in particular such method comprising steps (i)to (iv) as set forth in the previous paragraph, may be performed for asubject at two or more successive time points and the respectiveoutcomes at said successive time points may be compared, whereby thepresence or absence of a change between the prediction, diagnosis and/orprognosis of LVH, CF, PE or PAP at said successive time points isdetermined. The method thus allows to monitor a change in theprediction, diagnosis and/or prognosis of any one of LVH, CF, PE or PAPin a subject over time.

In an embodiment, a method for monitoring any one of LVH, CF, PE or PAPcomprises the steps of: (i) measuring the quantity of PERLECAN insamples from a subject from two or more successive time points; (ii)comparing the quantity of PERLECAN between the samples as measured in(i); (iii) finding a deviation or no deviation of the quantity ofPERLECAN between the samples as compared in (ii); and (iv) attributingsaid finding of deviation or no deviation to a change in LVH, CF, PE orPAP in the subject between the two or more successive time points. Themethod thus allows to monitor any one of LVH, CF, PE or PAP in a subjectover time.

Prediction or diagnosis of any one of LVH, CF, PE or PAP or a poorprognosis of LVH, CF, PE or PAP can in particular be associated with anelevated level of PERLECAN.

For example but without limitation, an elevated quantity (i.e., adeviation) of PERLECAN in a sample from a subject compared to areference value representing the prediction or diagnosis of no LVH, CF,PE or PAP (i.e., healthy state) or representing a good prognosis forLVH, CF, PE or PAP respectively indicates that the subject has or is atrisk of having LVH, CF, PE or PAP or indicates a poor prognosis for LVH,CF, PE or PAP in the subject.

Also disclosed is a method to determine whether a subject is or will beor is not or will not be (such as, for example, still is, or is nolonger) in need of a therapy to treat any one of LVH, CF, PE or PAP,comprising: (i) measuring the quantity of PERLECAN in the sample fromthe subject; (ii) comparing the quantity of PERLECAN measured in (i)with a reference value of the quantity of PERLECAN, said reference valuerepresenting a known diagnosis, prediction and/or prognosis of LVH, CF,PE or PAP; (iii) finding a deviation or no deviation of the quantity ofPERLECAN measured in (i) from said reference value; (iv) inferring fromsaid finding the presence or absence of a need for a therapy to treatLVH, CF, PE or PAP.

A therapy may be particularly indicated where steps (i) to (iii) allowfor a conclusion that the subject has or is at risk of having LVH, CF,PE or PAP or has a poor prognosis for LVH, CF, PE or PAP, such as forexample but without limitation, where the quantity of PERLECAN in thesample from the subject is elevated (i.e., a deviation) compared to areference value representing the prediction or diagnosis of no LVH, CF,PE or PAP (i.e., healthy state). Without limitation, a patient havingLVH, CF, PE or PAP upon admission to or during stay in a medical carecentre may be tested as taught herein for the necessity of continuing atreatment of said LVH, CF, PE or PAP, and may be discharged when suchtreatment is no longer needed or is needed only to a given limitedextent.

Any one prediction, diagnosis, prognosis and/or monitoring method astaught herein may preferably allow for sensitivity and/or specificity(preferably, sensitivity and specificity) of at least 50%, at least 60%,at least 70% or at least 80%, e.g., ≧85% or ≧90% or ≧95%, e.g., betweenabout 80% and 100% or between about 85% and 95%.

Reference throughout this specification to “diseases and/or conditions”encompasses any such diseases and conditions as disclosed herein insofarconsistent with the context of such a recitation, in particular butwithout limitation including renal dysfunction, dyspnea associated withor caused by renal failure, increased mortality of subjects havingdyspnea and/or acute heart failure and/or renal dysfunction, leftventricular hypertrophy, cardiac fibrosis, PE and PAP.

The present methods for predicting, diagnosing, prognosticating and/ormonitoring the diseases or conditions may be used in individuals whohave not yet been diagnosed as having such (for example, preventativescreening), or who have been diagnosed as having such, or who aresuspected of having such (for example, display one or morecharacteristic symptoms), or who are at risk of developing such (forexample, genetic predisposition; presence of one or more developmental,environmental or behavioural risk factors). The methods may also be usedto detect various stages of progression or severity of the diseases orconditions. The methods may also be used to detect response of thediseases or conditions to prophylactic or therapeutic treatments orother interventions. The methods can furthermore be used to help themedical practitioner in deciding upon worsening, status-quo, partialrecovery, or complete recovery of the patient from the diseases orconditions, resulting in either further treatment or observation or indischarge of the patient from medical care centre.

Any one of the herein described methods for predicting, diagnosing,prognosticating and/or monitoring the diseases or conditions may beemployed for population screening (such as, e.g., screening in a generalpopulation or in a population stratified based on one or more criteria,e.g., age, gender, ancestry, occupation, presence or absence of riskfactors of AHF, etc.). In any one the methods, the subject may form partof a patient population showing symptoms of dyspnea.

Diabetes and hypertension represent major risk factors for developingrenal dysfunction, more particularly (chronic) kidney failure. Hence,the present diagnosis, prediction, prognosis and/or monitoring methodsmay be preferably employed in such patients and patient populations,i.e., in subjects having or being at risk of having diabetes and/orhypertension (such as, e.g., in a screening setup).

The present methods enable the medical practitioner to monitor thedisease progress by measuring the level of PERLECAN in a sample of thepatient. For example, a decrease in PERLECAN level as compared to aprior PERLECAN level (e.g., at the time of the admission to ED)indicates the disease or condition in the subject is improving or hasimproved, while an increase of the PERLECAN level as compared to a priorPERLECAN level (e.g., at the time of the admission to ED) indicates thedisease or condition in the subject has worsened or is worsening. Suchworsening could possibly result in the recurrence of the disease orconditions.

In view of the present disclosure, also provided are:

-   -   the use of PERLECAN as a marker (biomarker);    -   the use of PERLECAN as a marker (biomarker) for any one disease        or condition as taught herein;    -   the use of PERLECAN for diagnosis, prediction, prognosis and/or        monitoring;    -   the use of PERLECAN for diagnosis, prediction, prognosis and/or        monitoring of any one disease or condition as taught herein;

particularly wherein said condition or disease may be chosen from renaldysfunction, dyspnea associated with or caused by renal failure,increased mortality of subjects having dyspnea and/or acute heartfailure and/or renal dysfunction, left ventricular hypertrophy, cardiacfibrosis, PE and PAP.

In the present prediction, diagnosis, prognosis and/or monitoringmethods the measurement of PERLECAN may also be combined with theassessment of one or more further biomarkers or clinical parametersrelevant for the respective diseases and conditions.

Consequently, also disclosed herein are methods, wherein the examinationphase of the methods further comprises measuring the presence or absenceand/or quantity of one or more such other markers in the sample from thesubject. In this respect, any known or yet unknown suitable marker couldbe used.

A reference throughout this specification to biomarkers “other thanPERLECAN” or “other biomarkers” generally encompasses such otherbiomarkers which are useful for predicting, diagnosing, prognosticatingand/or monitoring the diseases and conditions as disclosed herein. Bymeans of example and not limitation, biomarkers useful in evaluatingrenal dysfunction include creatinine (i.e., serum creatinine clearance),Cystatin C and neutrophil gelatinase-associated lipocalin (NGAL),beta-trace protein, kidney injury molecule 1 (KIM-1), interleukin-18(IL-18), and LTBP2. Further biomarkers useful in the present disclosureinclude inter alia B-type natriuretic peptide (BNP), pro-B-typenatriuretic peptide (proBNP), amino terminal pro-B-type natriureticpeptide (NTproBNP) and C-reactive peptide, and fragments or precursorsof any one thereof.

Hence, disclosed is a method for predicting, diagnosing and/orprognosticating the diseases or conditions as taught herein in a subjectcomprising the steps: (i) measuring the quantity of PERLECAN and thepresence or absence and/or quantity of said one or more other biomarkersin the sample from the subject; (ii) using the measurements of (i) toestablish a subject profile of the quantity of PERLECAN and the presenceor absence and/or quantity of said one or more other biomarkers; (iii)comparing said subject profile of (ii) to a reference profile of thequantity of PERLECAN and the presence or absence and/or quantity of saidone or more other biomarkers, said reference profile representing aknown prediction, diagnosis and/or prognosis of the conditions, symptomsand/or parameter values according to the invention; (iv) finding adeviation or no deviation of the subject profile of (ii) from thereference profile; (v) attributing said finding of deviation or nodeviation to a particular prediction, diagnosis and/or prognosis of therespective diseases or conditions in the subject.

Applying said method at two or more successive time points allows formonitoring the desired diseases or conditions.

The present methods may employ reference values for the quantity ofPERLECAN, which may be established according to known procedurespreviously employed for other biomarkers. Such reference values may beestablished either within (i.e., constituting a step of) or external to(i.e., not constituting a step of) the methods of the present inventionas defined herein. Accordingly, any one of the methods taught herein maycomprise a step of establishing a reference value for the quantity ofPERLECAN, said reference value representing either (a) a prediction ordiagnosis of the absence of the diseases or as taught herein or a goodprognosis thereof, or (b) a prediction or diagnosis of the diseases orconditions as taught herein or a poor prognosis thereof.

A further aspect provides a method for establishing a reference valuefor the quantity of PERLECAN, said reference value representing:

(a) a prediction or diagnosis of the absence of the diseases orconditions as taught herein or a good prognosis thereof, or

(b) a prediction or diagnosis of the diseases or conditions as taughtherein or a poor prognosis thereof, comprising:

(i) measuring the quantity of PERLECAN in:

-   -   (i a) one or more samples from one or more subjects not having        the respective diseases or conditions or not being at risk of        having such or having a good prognosis for such, or    -   (i b) one or more samples from one or more subjects having the        respective diseases or conditions or being at risk of having        such or having a poor prognosis for such, and

(ii) storing the quantity of PERLECAN

-   -   (ii a) as measured in (i a) as the reference value representing        the prediction or diagnosis of the absence of the respective        diseases or conditions or representing the good prognosis        therefore, or    -   (ii b) as measured in (i b) as the reference value representing        the prediction or diagnosis of the respective diseases or        conditions or representing the poor prognosis therefore.

The present methods may otherwise employ reference profiles for thequantity of PERLECAN and the presence or absence and/or quantity of oneor more other biomarkers, which may be established according to knownprocedures previously employed for other biomarkers. Such referenceprofiles may be established either within (i.e., constituting a step of)or external to (i.e., not constituting a step of) the present methods.Accordingly, the methods taught herein may comprise a step ofestablishing a reference profile for the quantity of PERLECAN and thepresence or absence and/or quantity of said one or more otherbiomarkers, said reference profile representing either (a) a predictionor diagnosis of the absence of the diseases or conditions as taughtherein or a good prognosis therefore, or (b) a prediction or diagnosisof the diseases or conditions as taught herein or a poor prognosistherefore.

A further aspect provides a method for establishing a reference profilefor the quantity of PERLECAN and the presence or absence and/or quantityof one or more other biomarkers useful for predicting, diagnosing,prognosticating and/or monitoring the diseases or conditions as taughtherein, said reference profile representing:

(a) a prediction or diagnosis of the absence of the respective diseasesor conditions or a good prognosis therefore, or

(b) a prediction or diagnosis of the respective diseases or conditionsor a poor prognosis therefore, comprising:

(i) measuring the quantity of PERLECAN and the presence or absenceand/or quantity of said one or more other biomarkers in:

-   -   (i a) one or more samples from one or more subjects not having        the respective diseases or conditions or not being at risk of        having such or having a good prognosis for such; or    -   (i b) one or more samples from one or more subjects having the        respective diseases or conditions or being at risk of having        such or having a poor prognosis for such;

(ii)

-   -   (ii a) using the measurements of (i a) to create a profile of        the quantity of PERLECAN and the presence or absence and/or        quantity of said one or more other biomarkers; or    -   (ii b) using the measurements of (i b) to create a profile of        the quantity of PERLECAN and the presence or absence and/or        quantity of said one or more other biomarkers;    -   (iii a) storing the profile of (ii a) as the reference profile        representing the prediction or diagnosis of the absence of the        respective diseases or conditions or representing the good        prognosis therefore; or    -   (iii b) storing the profile of (ii b) as the reference profile        representing the prediction or diagnosis of the respective        diseases conditions or representing the poor prognosis        therefore.

Further provided is a method for establishing a PERLECAN base-line orreference value in a subject, comprising: (i) measuring the quantity ofPERLECAN in the sample from the subject at different time points whereinthe subject is not suffering from the diseases or conditions as taughtherein, and (ii) calculating the range or mean value of the subject,which is the PERLECAN base-line or reference value for said subject.

Preferably, the subject as intended in any one of the present methodsmay be human.

The quantity of PERLECAN and/or the presence or absence and/or quantityof the one or more other biomarkers may be measured by any suitabletechnique such as may be known in the art. For example, the quantity ofPERLECAN and/or the presence or absence and/or quantity of the one ormore other biomarkers may be measured using, respectively, a bindingagent capable of specifically binding to PERLECAN and/or to fragmentsthereof, and a binding agent capable of specifically binding to said oneor more other biomarkers. For example, the binding agent may be anantibody, aptamer, photoaptamer, protein, peptide, peptidomimetic or asmall molecule. For example, the quantity of PERLECAN and/or thepresence or absence and/or quantity of the one or more other biomarkersmay be measured using an immunoassay technology or a mass spectrometryanalysis method or a chromatography method, or a combination of saidmethods.

Further disclosed is a kit for predicting, diagnosing, prognosticatingand/or monitoring the diseases or conditions as taught herein in asubject, the kit comprising (i) means for measuring the quantity ofPERLECAN in a sample from the subject, and optionally and preferably(ii) a reference value of the quantity of PERLECAN or means forestablishing said reference value, wherein said reference valuerepresents a known prediction, diagnosis and/or prognosis of therespective diseases or conditions. The kit thus allows one to: measurethe quantity of PERLECAN in the sample from the subject by means (i);compare the quantity of PERLECAN measured by means (i) with thereference value of (ii) or established by means (ii); find a deviationor no deviation of the quantity of PERLECAN measured by means (i) fromthe reference value of (ii); and consequently attribute said finding ofdeviation or no deviation to a particular prediction, diagnosis and/orprognosis of the respective diseases or conditions in the subject.

A further embodiment provides a kit for predicting, diagnosing,prognosticating and/or monitoring the diseases or conditions as taughtherein in a subject, the kit comprising (i) means for measuring thequantity of PERLECAN in a sample from the subject and (ii) means formeasuring the presence or absence and/or quantity of one or more otherbiomarkers in the sample from the subject, and optionally and preferably(iii) means for establishing a subject profile of the quantity ofPERLECAN and the presence or absence and/or quantity of said one or moreother biomarkers, and optionally and preferably (iv) a reference profileof the quantity of PERLECAN and the presence or absence and/or quantityof said one or more other biomarkers, or means for establishing saidreference profile, said reference profile representing a knownprediction, diagnosis and/or prognosis of the conditions, symptomsand/or parameter values according to the invention. Such kit thus allowsone to: measure the quantity of PERLECAN and the presence or absenceand/or quantity of said one or more other biomarkers in the sample fromthe subject by respectively means (i) and (ii); establish (e.g., usingmeans included in the kit or using suitable external means) a subjectprofile of the quantity of PERLECAN and the presence or absence and/orquantity of said one or more other biomarkers based on saidmeasurements; compare the subject profile with the reference profile of(iv) or established by means (iv); find a deviation or no deviation ofsaid subject profile from said reference profile; and consequentlyattribute said finding of deviation or no deviation to a particularprediction, diagnosis and/or prognosis of the respective diseases orconditions in the subject.

The means for measuring the quantity of PERLECAN and/or the presence orabsence and/or quantity of the one or more other biomarkers in thepresent kits may comprise, respectively, one or more binding agentscapable of specifically binding to PERLECAN and/or to fragments thereof,and one or more binding agents capable of specifically binding to saidone or more other biomarkers. For example, any one of said one or morebinding agents may be an antibody, aptamer, photoaptamer, protein,peptide, peptidomimetic or a small molecule. For example, any one ofsaid one or more binding agents may be advantageously immobilised on asolid phase or support. The means for measuring the quantity of PERLECANand/or the presence or absence and/or quantity of the one or more otherbiomarkers in the present kits may employ an immunoassay technology ormass spectrometry analysis technology or chromatography technology, or acombination of said technologies.

Disclosed is thus also a kit for predicting, diagnosing, prognosticatingand/or monitoring the diseases or conditions as taught hereincomprising: (a) one or more binding agents capable of specificallybinding to PERLECAN and/or to fragments thereof; (b) preferably, a knownquantity or concentration of PERLECAN and/or a fragment thereof (e.g.,for use as controls, standards and/or calibrators); (c) preferably, areference value of the quantity of PERLECAN, or means for establishingsaid reference value. Said components under (a) and/or (c) may besuitably labelled as taught elsewhere in this specification.

Also disclosed is a kit for predicting, diagnosing and/orprognosticating the diseases or conditions as taught herein comprising:(a) one or more binding agents capable of specifically binding toPERLECAN and/or to fragments thereof; (b) one or more binding agentscapable of specifically binding to one or more other biomarkers; (c)preferably, a known quantity or concentration of PERLECAN and/or afragment thereof and a known quantity or concentration of said one ormore other biomarkers (e.g., for use as controls, standards and/orcalibrators); (d) preferably, a reference profile of the quantity ofPERLECAN and the presence or absence and/or quantity of said one or moreother biomarkers, or means for establishing said reference profiles.Said components under (a), (b) and/or (c) may be suitably labelled astaught elsewhere in this specification.

Further disclosed is the use of the kit as described herein fordiagnosing, predicting, prognosticating and/or monitoring the diseasesor conditions as taught herein.

Also disclosed are reagents and tools useful for measuring PERLECAN andoptionally the one or more other biomarkers concerned herein.

Hence, disclosed is a protein, polypeptide or peptide array ormicroarray comprising (a) PERLECAN and/or a fragment thereof, preferablya known quantity or concentration of said PERLECAN and/or fragmentthereof; and (b) optionally and preferably, one or more otherbiomarkers, preferably a known quantity or concentration of said one ormore other biomarkers.

Also disclosed is a binding agent array or microarray comprising: (a)one or more binding agents capable of specifically binding to PERLECANand/or to fragments thereof, preferably a known quantity orconcentration of said binding agents; and (b) optionally and preferably,one or more binding agents capable of specifically binding to one ormore other biomarkers, preferably a known quantity or concentration ofsaid binding agents.

Also disclosed are kits as taught here above configured as portabledevices, such as, for example, bed-side devices, for use at home or inclinical settings.

A related aspect thus provides a portable testing device capable ofmeasuring the quantity of PERLECAN in a sample from a subjectcomprising: (i) means for obtaining a sample from the subject, (ii)means for measuring the quantity of PERLECAN in said sample, and (iii)means for visualising the quantity of PERLECAN measured in the sample.

In an embodiment, the means of parts (ii) and (iii) may be the same,thus providing a portable testing device capable of measuring thequantity of PERLECAN in a sample from a subject comprising (i) means forobtaining a sample from the subject; and (ii) means for measuring thequantity of PERLECAN in said sample and visualising the quantity ofPERLECAN measured in the sample.

In an embodiment, said visualising means is capable of indicatingwhether the quantity of PERLECAN in the sample is above or below acertain threshold level and/or whether the quantity of PERLECAN in thesample deviates or not from a reference value of the quantity ofPERLECAN, said reference value representing a known prediction,diagnosis and/or prognosis of the diseases or conditions as taughtherein. Hence, the portable testing device may suitably also comprisesaid reference value or means for establishing the reference value.

In an embodiment, the threshold level is chosen such that the quantityof PERLECAN in the sample above said threshold level indicates that thesubject has or is at risk of having the respective disease or conditionor indicates a poor prognosis for such in the subject, and the quantityof PERLECAN in the sample below said threshold level indicates that thesubject does not have or is not at risk of having the diseases orconditions as taught herein or indicates a good prognosis for such inthe subject.

In an embodiment, the portable testing device comprises a referencevalue representing the prediction or diagnosis of the absence of thediseases or conditions as taught herein or representing a good prognosisfor such, or comprises means for establishing said reference value, andan elevated quantity of PERLECAN in the sample from the subject comparedto said reference value indicates that the subject has or is at risk ofhaving the respective disease or condition or indicates a poor prognosisfor such in the subject. In another embodiment, the portable testingdevice comprises a reference value representing the prediction ordiagnosis of the diseases or conditions as taught herein or representinga poor prognosis for such, or comprises means for establishing saidreference value, and a comparable quantity of PERLECAN in the samplefrom the subject compared to said reference value indicates that thesubject has or is at risk of having the respective disease or conditionor indicates a poor prognosis for such in the subject.

In a further embodiment, the measuring (and optionally visualisation)means of the portable testing device may comprise a solid support havinga proximal and distal end, comprising: —a sample application zone in thevicinity of the proximal end; —a reaction zone distal to the sampleapplication zone; and—a detection zone distal to the reaction zone;—optionally control standards comprising PERLECAN protein or peptidefragments, whereby said support has a capillary property that directs aflow of fluid sample applied in the application zone in a direction fromthe proximal end to the distal end; and—optionally comprising a fluidsource improving the capillary flow of a more viscous sample.

The reaction zone may comprise one or more bands of a PERLECAN-specificbinding molecules conjugated to a detection agent, which PERLECANspecific binding molecule conjugate is disposed on the solid supportsuch that it can migrate with the capillary flow of fluid; and whereinthe detection zone comprises one or more capture bands comprising apopulation of PERLECAN specific molecule immobilised on the solidsupport.

The reaction zone may additionally comprise one or more bands of capturePERLECAN-specific binding molecules in an amount sufficient to prevent athreshold quantity of PERLECAN specific binding molecule conjugates tomigrate to the detection zone. Alternatively, said device additionallycomprises means for comparing the amount of captured PERLECAN specificbinding molecule conjugate with a threshold value.

Other aspects relate to the realisation that PERLECAN may be a valuabletarget for therapeutic and/or prophylactic interventions in diseases andconditions as taught herein, in particular but without limitationincluding renal dysfunction, dyspnea associated with or caused by renalfailure, increased mortality of subjects having dyspnea and/or acuteheart failure and/or renal dysfunction, left ventricular hypertrophy,cardiac fibrosis, PE and PAP.

Hence, also disclosed herein are any one and all of the following:

(1) an agent that is able to modulate the level and/or the activity ofPERLECAN for use as a medicament, preferably for use in the treatment ofany one disease or condition as taught herein;

(2) use of an agent that is able to modulate the level and/or theactivity of PERLECAN for the manufacture of a medicament for thetreatment of any one disease or condition as taught herein; or use of anagent that is able to modulate the level and/or the activity of PERLECANfor the treatment of any one disease or condition as taught herein;

(3) a method for treating any one disease or condition as taught hereinin a subject in need of such treatment, comprising administering to saidsubject a therapeutically or prophylactically effective amount of anagent that is able to modulate the level and/or the activity ofPERLECAN;

(4) The subject matter as set forth in any one of (1) to (3) above,wherein the agent is able to reduce or increase the level and/or theactivity of PERLECAN, preferably to reduce the level and/or the activityof PERLECAN.

(5) The subject matter as set forth in any one of (1) to (4) above,wherein said agent is able to specifically bind to PERLECAN.

(6) The subject matter as set forth in any one of (1) to (5) above,wherein said agent is an antibody or a fragment or derivative thereof; apolypeptide; a peptide; a peptidomimetic; an aptamer; a photoaptamer; ora chemical substance, preferably an organic molecule, more preferably asmall organic molecule.

(7) The subject matter as set forth in any one of (1) to (4) above,wherein the agent is able to reduce or inhibit the expression ofPERLECAN, preferably wherein said agent is an antisense agent; aribozyme; or an agent capable of causing RNA interference.

(8) The subject matter as set forth in any one of (1) to (4) above,wherein said agent is able to reduce or inhibit the level and/oractivity of PERLECAN, preferably wherein said agent is a recombinant orisolated deletion construct of the PERLECAN polypeptide having adominant negative activity over the native PERLECAN.

(9) An assay to select, from a group of test agents, a candidate agentpotentially useful in the treatment of any one disease or condition astaught herein, said assay comprising determining whether a tested agentcan modulate, such as increase or reduce and preferably reduce, thelevel and/or activity of PERLECAN.

(10) The assay as set forth in (9) above, further comprising use of theselected candidate agent for the preparation of a composition foradministration to and monitoring the prophylactic and/or therapeuticeffect thereof in a non-human animal model, preferably a non-humanmammal model, of any one disease or condition as taught herein.

(11) The agent isolated by the assay as set forth in (10) above.

(12) A pharmaceutical composition or formulation comprising aprophylactically and/or therapeutically effective amount of one or moreagents as set forth in any one of (1) to (8) or (10) above, or apharmaceutically acceptable N-oxide form, addition salt, prodrug orsolvate thereof, and further comprising one or more of pharmaceuticallyacceptable carriers.

(13) A method for producing the pharmaceutical composition orformulation as set forth in (12) above, comprising admixing said one ormore agents with said one or more pharmaceutically acceptable carriers.

Said condition or disease as set forth in any one of (1) to (13) abovemay be particularly chosen from renal dysfunction, dyspnea associatedwith or caused by renal failure, increased mortality of subjects havingdyspnea and/or acute heart failure and/or renal dysfunction, leftventricular hypertrophy, cardiac fibrosis, PE and PAP.

Also contemplated is thus a method (a screening assay) for selecting anagent capable of specifically binding to PERLECAN (e.g., gene orprotein) comprising: (a) providing one or more, preferably a pluralityof, test PERLECAN-binding agents; (b) selecting from the testPERLECAN-binding agents of (a) those which bind to PERLECAN; and (c)counter-selecting (i.e., removing) from the test PERLECAN-binding agentsselected in (b) those which bind to any one or more other, unintended orundesired, targets.

Alternatively, one could envisage an inhibitor of the moleculesresponsible for the processing of the PERLECAN molecule or fragmentthereof such as inhibitors of the Cathepsin L enzyme, which has beenshown to cleave the LG3 fragment from the endorepellin fragment ofPERLECAN. Some CathL inhibitors are commercially available. Non-limitingexamples are: ZFF-FMK (Calbiochem) and ZFA-FMK (MP Biomedicals), whichare irreversible and cell-permeable CathL inhibitors.

Binding between test PERLECAN-binding agents and PERLECAN may beadvantageously tested by contacting (i.e., combining, exposing orincubating) said PERLECAN with the test PERLECAN-binding agents underconditions generally conducive for such binding. For example and withoutlimitation, binding between test PERLECAN-binding agents and thePERLECAN may be suitably tested in vitro; or may be tested in host cellsor host organisms comprising the PERLECAN and exposed to or configuredto express the test PERLECAN-binding agents.

Without limitation, the PERLECAN-binding or PERLECAN-modulating agentsmay be capable of binding PERLECAN or modulating the activity and/orlevel of the PERLECAN in vitro, in a cell, in an organ and/or in anorganism.

In the screening assays as set forth in any one of (9) and (10) above,modulation of the activity and/or level of the PERLECAN by testPERLECAN-modulating agents may be advantageously tested by contacting(i.e., combining, exposing or incubating) said PERLECAN (e.g., gene orprotein) with the test PERLECAN-modulating agents under conditionsgenerally conducive for such modulation. By means of example and notlimitation, where modulation of the activity and/or level of thePERLECAN results from binding of the test PERLECAN-modulating agents tothe PERLECAN, said conditions may be generally conducive for suchbinding. For example and without limitation, modulation of the activityand/or level of the PERLECAN by test PERLECAN-modulating agents may besuitably tested in vitro; or may be tested in host cells or hostorganisms comprising the LPBT2 and exposed to or configured to expressthe test PERLECAN-modulating agents.

As well contemplated are:

-   -   PERLECAN for use as a medicament, preferably for use in the        treatment of any one disease or condition as taught herein;    -   use of PERLECAN for the manufacture of a medicament for the        treatment of any one disease or condition as taught herein;    -   use of PERLECAN for the treatment of any one disease or        condition as taught herein;    -   a method for treating any one disease or condition as taught        herein in a subject in need of such treatment, comprising        administering to said subject a therapeutically or        prophylactically effective amount of PERLECAN;    -   particularly wherein said condition or disease may be chosen        from renal dysfunction, dyspnea associated with or caused by        renal failure, increased mortality of subjects having dyspnea        and/or acute heart failure and/or renal dysfunction, left        ventricular hypertrophy, cardiac fibrosis, PE and PAP.

These and further aspects and preferred embodiments are described in thefollowing sections and in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates sequences of full length PERLECAN (SEQ ID NO. 1). Theunderlined part depicts the endorepellin domain (SEQ ID NO. 2) and thebold underlined part depicts the LG-3 domain (SEQ ID NO. 3). Thepeptides detected by the MASSTERCLASS™ or MASStermind™ technology aredouble underligned (SEQ ID No's 4 and 5 respectively).

FIG. 2 illustrates correlation of PERLECAN levels with estimatedglomerular filtration rate (eGFR) and Cystatin C levels in all 154patients.

FIG. 3 illustrates that PERLECAN shows comparable performance toCystatin C in discriminating patients with reduced eGFR (herein <60ml/min/1.73 m²) from patients with normal eGFR. Receiver operatingcharacteristic curve of Cystatin C (dark grey) compared to PERLECAN(light grey). Calculated median area under the curve (AUC) and 95%confidence intervals are for Cystatin C: 0.94 (0.90-0.96) and forPERLECAN: 0.91 (0.83-0.97).

FIG. 4 shows box and whisker plots for PERLECAN in patients with reduced(<60) and normal (>90) and intermediate eGFR (60-90).

FIG. 5 shows a graph of the one year mortality prediction of the Basel Vcohort, correlated with the levels of different renal failure markers.From the graph it is clear that PERLECAN is correlated to mortality in asimilar manner as the BNP, NT-pro-BNP and Cystatin-C markers.

FIG. 6 illustrates the performance of PERLECAN as predictive and earlydiagnostic marker for renal replacement therapy requirement incomparison to creatinine based measures. From the graph it is clear thatPERLECAN is a better and earlier predictor than creatinine and eGfr.

FIG. 7: FIG. 7A shows a schematic overview of PERLECAN and its derivedactive peptides endorepellin and LG3 as well as the different peptidesused for MASSTERCLASS quantitation and the correlation of MASSTERCLASSread-outs based on Seq ID No: 7 with eGfr. FIG. 7B shows the correlationof MASSTERCLASS read-outs based on Seq ID Nos: 4 and 6 with eGfr.

DETAILED DESCRIPTION

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The term “about” as used herein when referring to a measurable valuesuch as a parameter, an amount, a temporal duration, and the like, ismeant to encompass variations of and from the specified value, inparticular variations of +/−10% or less, preferably +/−5% or less, morepreferably +/−1% or less, and still more preferably +/−0.1% or less ofand from the specified value, insofar such variations are appropriate toperform in the disclosed invention. It is to be understood that thevalue to which the modifier “about” refers is itself also specifically,and preferably, disclosed.

All documents cited in the present specification are hereby incorporatedby reference in their entirety.

Unless otherwise specified, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions may be includedto better appreciate the teaching of the present invention.

The inventors show that PERLECAN is a valuable biomarker particularlyfor renal (dys)function and mortality in subjects having dyspnea and/oracute heart failure and/or renal dysfunction, and further for leftventricular hypertrophy, cardiac fibrosis, preeclampsia (PE) andpregnancy-associated proteinuria (PAP).

The term “biomarker” is widespread in the art and may broadly denote abiological molecule and/or a detectable portion thereof whosequalitative and/or quantitative evaluation in a subject is predictive orinformative (e.g., predictive, diagnostic and/or prognostic) withrespect to one or more aspects of the subject's phenotype and/orgenotype, such as, for example, with respect to the status of thesubject as to a given disease or condition.

Reference herein to “disease(s) and/or condition(s) as taught herein” ora similar reference encompasses any such diseases and conditions asdisclosed herein insofar consistent with the context of such arecitation, in particular but without limitation including renaldysfunction, dyspnea associated with or caused by renal failure,increased mortality of subjects having dyspnea and/or acute heartfailure and/or renal dysfunction, left ventricular hypertrophy, cardiacfibrosis, PE, proteinuria, e.g. proteinuria associated with pregnancy(PAP), or with etabolic syndrome or with Type II diabetes.

Renal or kidney dysfunction, which may also be interchangeably known asrenal or kidney failure or insufficiency, generally encompasses states,diseases and conditions in which the functioning of renal tissue isinadequate, particularly wherein kidney excretory function iscompromised.

Signs and symptoms of renal dysfunction may include without limitationany one or more of increased levels of urea and/or nitrogen in theblood; lower than normal creatinine clearance and higher than normalcreatinine levels in blood; lower than normal free water clearance;

volume overload and swelling; abnormal acid levels; higher than normallevels of potassium, calcium and/or phosphate in blood; changes inurination (e.g., volume, osmolarity); microalbuminuria ormacroalbuminuria; altered activity of kidney enzymes such as gammaglutamyl synthetase; fatigue; skin rash or itching; nausea; dyspnea;reduced kidney size; haematuria and anaemia.

Conventionally, renal dysfunction is deemed as comprising major classesdenoted as acute renal or kidney failure (acute renal or kidney diseaseor injury, e.g., acute kidney injury or “AKI”) or chronic renal orkidney failure (chronic renal or kidney disease). Whereas progression istypically fast (e.g., days to weeks) in acute renal failure, renalfailure may be traditionally regarded as chronic if it persists for atleast 3 months and its progression may take in the range of years.

Acute renal dysfunction or failure may be staged (classified, graded)into 5 distinct stages using the “RIFLE” (Risk, Injury, Failure, Loss,end-stage renal disease) staging system as set out here below (based onLameire et al. 2005, Lancet 365: 417-430):

GFR (based on serum creatinine) criteria Stage GFR = glomerularfiltration rate Urine output criteria “Risk” Serum creatinine increased1.5 times <0.5 mL/kg/h for 6 h “Injury” Serum creatinine increased 2.0times <0.5 mL/kg/h for 12 h “Failure” Serum creatinine increased 3.0times, <0.3 mL/kg/h for 24 h or creatinine >355 mM/L when there oranuria for 12 h was an acute rise of >44 mM/L “Loss” Persistent acuterenal failure — >4 weeks “End- End-stage renal disease >3 months —stage”

Chronic renal dysfunction or failure may be staged (classified, graded)based on GFR as set out here below (based on Levey et al. 2005, KidneyInt 67: 2089-2100):

Stage 1: GFR 90 mL/min (normal or elevated GFR)

Stage 2: GFR=60-89 mL/min (mild GFR reduction)

Stage 3: GFR=30-59 mL/min (moderate GFR reduction)

Stage 4: GFR=15-29 mL/min (severe GFR reduction)

Stage 5: GFR<15 mL/min (renal failure)

Other staging methods for renal failure resulting in similar orcomparable classifications of different stages of renal failure may beused herein.

The present diagnosis, prediction, prognosis and/or monitoring methodsmay allow to determine that a subject has or is at risk of having acuteor chronic renal failure, such as in particular determine any one of theabove-described or comparable stages of acute or chronic renal failurein the subject, and/or may allow to discriminate between said stages inthe subject.

The causes of acute renal deterioration may be pre-renal, post-renaland/or intra-renal. Pre-renal causes include lack of sufficient bloodsupply to the kidneys (i.e., renal hypoperfusion), which in turn may becaused by inter alia haemorrhage, massive blood loss, congestive heartfailure, decompensated liver cirrhosis (liver cirrhosis withcomplications such as bleedings, ascites), damaged kidney blood vessels,sepsis or systemic inflammation due to infection. Patients thatunderwent a surgical or operative treatment sometimes suffer from acutekidney injury (AKI), resulting in a temporary loss or reduction of thefunction of the kidneys. Post-renal causes include obstructions of urinecollection systems or extra-renal drainage (i.e., obstructive uropathy),which in turn may be caused by inter alia medication interfering withnormal bladder emptying, prostate diseases, kidney stones, abdominalmalignancy (such as ovarian cancer or colorectal cancer), or obstructedurinary catheter. Intra-renal causes include renal tissue-destroyingconditions, such as vasculitis, malignant hypertension, acuteglomerulonephritis, acute interstitial nephritis and acute tubularnecrosis. They can be caused without limitation by ischemic events (suchas, e.g., haemoglobinuria, myoglobinuria and myoloma) or by nephrotoxicsubstances (such as, e.g., antibiotics, radio contrast agents, uricacid, oxalate and drug induced renal toxicity). Subjects having or beingat risk of having the above states, conditions or diseases may have ormay be at risk of developing acute renal failure. Hence, the presentdiagnosis, prediction, prognosis and/or monitoring methods may bepreferably employed in such patients.

Causes of chronic renal deterioration may include inter alia vasculardiseases, such as, e.g., bilateral renal artery stenosis, ischemicnephropathy, haemolytic-uremic syndrome and vasculitis, and furtherfocal segmental nephrosclerosis, glomerulosclerosis, glomerulonephritis,IgA nephritis, diabetic nephropathy, lupus nephritis, polycystic kidneydisease, chronic tubulointerstitial nephritis (e.g., drug and/ortoxin-induced), renal fibrosis, kidney stones, and prostate diseases.Subjects having or being at risk of having the above states, conditionsor diseases may have or may be at risk of developing chronic renalfailure. Proteinuria, essentially the abnormal presence of proteins inthe urine of a subject, can be a sign of renal dysfunction and occurs inmany disorders such nephrotic syndromes, nephropathys, glomerulardiseases, such as membranous glomerulonephritis, focal segmentalglomerulonephritis, minimal change disease (lipoid nephrosis),Eeclampsia or Preclampsia (PE) and Pregnancy associated Proteinuria(PAP), Type II diabetes etc. Type II diabetes develops inobese/metabolic syndrome patients. These patients typically developglomerulus damage (inflammation, hypertension, metabolic changes etc.)to end up with a reduced GFR and proteinuria. Very typical for thesepatients is that the reduced GFR is preceded by a hyperfiltration phase.

Postoperative patients are prone to develop kidney related problems,mostly apparent as acute kidney injury (AKI). Further a significantpercentage of these patients require haemodialysis or renal replacementtherapy (RRT) in the days following surgery, this to attain soluteclearance and fluid balance while waiting for kidney function torecover. Timely institution of RRT is fundamental to achieve this goal.When no supportive therapy is started timely, the kidneys may sufferpermanent damage and not recover fully. Current indications for RRTinclude persistent hyperkalemia, severe acidosis and hypervolemia thatare unresponsive to conservative measures. Early RRT is used to describethe initiation of dialysis therapy before nitrogenous waste productsreach some arbitrarily predefined “critical” blood value, irrespectiveof clinical indications. A meta-analysis shows that early institution ofRRT might be associated with improved outcomes (i.e. decreasedmortality) in patients with AKI (Seabra et al., 2008; American Journalof Kidney diseases; Vol 52 (2), 272-284). Clearly, when a patients isput on RRT before the traditional creatinine or urea based time pointsurvival is improved. While creatinine or urea levels are currently usedfor deciding upon starting RRT, there is currently no good criterion forearly RRT intervention. There is no objective early measure for the needof early RRT initiation and a biomarker that can specifically identifythose patients that will benefit from early RRT is warranted.

The present invention provides for a new biomarker, PERLECAN, especiallyits endorepellin region, more in particular its LG3 region, for theearly establishment as to whether a patient requires RRT.

Hence, the present diagnosis, prediction, prognosis and/or monitoringmethods may be preferably employed preferably in the types of patientslisted above.

Dyspnea (dyspnoea or shortness of breath) is known per se and mayparticularly refer to a common and distressing symptom experienced bysubjects as unpleasant or uncomfortable respiratory sensations, that maybe more particularly defined as a “subjective experience of breathingdiscomfort that consists of qualitatively distinct sensations that varyin intensity”. Dyspnea may be connected to a range of underlyingpathologies.

The terms “heart failure”, “acute heart failure” and “chronic heartfailure” as used herein carry their respective art-established meanings.By means of further guidance, the term “heart failure” as used hereinbroadly refers to pathological conditions characterised by an impaireddiastolic or systolic blood flow rate and thus insufficient blood flowfrom the ventricle to peripheral organs.

“Acute heart failure” or also termed “acute decompensated heart failure”may be defined as the rapid onset of symptoms and signs secondary toabnormal cardiac function, resulting in the need for urgent therapy. AHFcan present itself acute de novo (new onset of acute heart failure in apatient without previously known cardiac dysfunction) or as acutedecompensation of CHF.

The cardiac dysfunction may be related to systolic or diastolicdysfunction, to abnormalities in cardiac rhythm, or to preload andafterload mismatch. It is often life threatening and requires urgenttreatment. According to established classification, AHF includes severaldistinct clinical conditions of presenting patients: (I) acutedecompensated congestive heart failure, (II) AHF withhypertension/hypertensive crisis, (III) AHF with pulmonary oedema, (IVa)cardiogenic shock/low output syndrome, (IVb) severe cardiogenic shock,(V) high output failure, and (VI) right-sided acute heart failure. Fordetailed clinical description, classification and diagnosis of AHF, andfor summary of further AHF classification systems including the Killipclassification, the Forrester classification and the ‘clinical severity’classification, refer inter alia to Nieminen et al. 2005 (“Executivesummary of the guidelines on the diagnosis and treatment of acute heartfailure: the Task Force on Acute Heart Failure of the European Societyof Cardiology”. Eur Heart J 26: 384-416) and references therein.

The term “chronic heart failure” (CHF) generally refers to a case ofheart failure that progresses so slowly that various compensatorymechanisms work to bring the disease into equilibrium. Common clinicalsymptoms of CHF include inter alia any one or more of breathlessness,diminishing exercise capacity, fatigue, lethargy and peripheral oedema.Other less common symptoms include any one or more of palpitations,memory or sleep disturbance and confusion, and usually co-occur with oneor more of the above recited common symptoms.

Left ventricular hypertrophy (LVH) generally encompasses the thickeningof the myocardium of the left ventricle of the heart. LVH may representa pathological reaction to cardiovascular diseases that increase theafterload (e.g., aortic stenosis or aortic insufficiency) or high bloodpressure. LVH may also represent primary hypertrophic cardiomyopathy.LVH diagnosis may be made inter alia using echocardiography, usingcriteria known per se such as the Sokolow-Lyon index, the Cornellvoltage criteria, the Romhilt-Estes point score system or othervoltage-based criteria.

Cardiac fibrosis generally encompasses abnormal thickening of the heartvalves due to inappropriate proliferation of cardiac fibroblasts and theconcomitant excessive production of matrix proteins.

By “preeclampsia” (PE or pre-eclampsia) is meant the multi-systemdisorder that is characterised by hypertension with proteinuria oroedema, or both, glomerular dysfunction, brain oedema, liver oedema, orcoagulation abnormalities due to pregnancy or the influence of a recentpregnancy and all complications associated with the disorder.Pre-eclampsia generally occurs after the 20th week of gestation.Pre-eclampsia is generally defined as some combination of the followingsymptoms: (1) a systolic blood pressure (BP)>140 mmHg and a diastolicBP>90 mmHg after 20 weeks gestation (generally measured on twooccasions, 4-168 hours apart), (2) new onset proteinuria (1+ by dipstickon urinanalysis, >300 mg of protein in a 24-hour urine collection, or asingle random urine sample having a protein/creatinine ratio >0.3), and(3) resolution of hypertension and proteinuria by 12 weeks postpartum.

Severe pre-eclampsia is generally defined as (1) a diastolic BP>110 mmHg(generally measured on two occasions, 4-168 hours apart) or (2)proteinuria characterised by a measurement of 3.5 g or more protein in a24-hour urine collection or two random urine specimens with at least 3+protein by dipstick. In pre-eclampsia, hypertension and proteinuriagenerally occur within seven days of each other. In severepre-eclampsia, severe hypertension, severe proteinuria and HELLPsyndrome (haemolysis, elevated liver enzymes, low platelets) oreclampsia can occur simultaneously or only one symptom at a time.Occasionally, severe pre-eclampsia can lead to the development ofseizures. This severe form of the syndrome is referred to as“eclampsia.” Eclampsia can also include dysfunction or damage to severalorgans or tissues such as the liver (e.g., hepatocellular damage,periportal necrosis) and the central nervous system (e.g., cerebraloedema and cerebral haemorrhage). The aetiology of the seizures isthought to be secondary to the development of cerebral oedema and focalspasm of small blood vessels in the kidney. Preeclampsia is associatedwith foetal complications such as intrauterine growth retardation (IUGR)and small for gestational age (SGA). By “small for gestational age(SGA)” is meant a foetus whose birth weight is a weight less than 2,500gm or below the 10th percentile for gestational age according to U.S.tables of birth weight for gestational age by race, parity, and infantsex as defined by World Health Organization (WHO) (Zhang and Bowes 1995,Obstet Gynecol 86: 200-208).

The terms “predicting” or “prediction”, “diagnosing” or “diagnosis” and“prognosticating” or “prognosis” are commonplace and well-understood inmedical and clinical practice. It shall be understood that the phrase “amethod for predicting, diagnosing and/or prognosticating” a givendisease or condition may also be interchanged with phrases such as “amethod for prediction, diagnosis and/or prognosis” of said disease orcondition or “a method for making (or determining or establishing) aprediction, diagnosis and/or prognosis” of said disease or condition, orthe like.

By means of further explanation and without limitation, “predicting” or“prediction” generally refer to an advance declaration, indication orforetelling of a disease or condition in a subject not (yet) having saiddisease or condition. For example, a prediction of a disease orcondition in a subject may indicate a probability, chance or risk thatthe subject will develop said disease or condition, for example within acertain time period or by a certain age. Said probability, chance orrisk may be indicated inter alia as an absolute value, range orstatistics, or may be indicated relative to a suitable control subjector subject population (such as, e.g., relative to a general, normal orhealthy subject or subject population). Hence, the probability, chanceor risk that a subject will develop a disease or condition may beadvantageously indicated as increased or decreased, or as fold-increasedor fold-decreased relative to a suitable control subject or subjectpopulation. As used herein, the term “prediction” of the conditions ordiseases as taught herein in a subject may also particularly mean thatthe subject has a ‘positive’ prediction of such, i.e., that the subjectis at risk of having such (e.g., the risk is significantly increasedvis-à-vis a control subject or subject population). The term “predictionof no” diseases or conditions as taught herein as described herein in asubject may particularly mean that the subject has a ‘negative’prediction of such, i.e., that the subject's risk of having such is notsignificantly increased vis-à-vis a control subject or subjectpopulation.

The terms “diagnosing” or “diagnosis” generally refer to the process oract of recognising, deciding on or concluding on a disease or conditionin a subject on the basis of symptoms and signs and/or from results ofvarious diagnostic procedures (such as, for example, from knowing thepresence, absence and/or quantity of one or more biomarkerscharacteristic of the diagnosed disease or condition). As used herein,“diagnosis of” the diseases or conditions as taught herein in a subjectmay particularly mean that the subject has such, hence, is diagnosed ashaving such. “Diagnosis of no” diseases or conditions as taught hereinin a subject may particularly mean that the subject does not have such,hence, is diagnosed as not having such. A subject may be diagnosed asnot having such despite displaying one or more conventional symptoms orsigns reminiscent of such.

The terms “prognosticating” or “prognosis” generally refer to ananticipation on the progression of a disease or condition and theprospect (e.g., the probability, duration, and/or extent) of recovery.

A good prognosis of the diseases or conditions taught herein maygenerally encompass anticipation of a satisfactory partial or completerecovery from the diseases or conditions, preferably within anacceptable time period. A good prognosis of such may more commonlyencompass anticipation of not further worsening or aggravating of such,preferably within a given time period.

A poor prognosis of the diseases or conditions as taught herein maygenerally encompass anticipation of a substandard recovery and/orunsatisfactorily slow recovery, or to substantially no recovery or evenfurther worsening of such.

The term “subject” or “patient” as used herein typically denotes humans,but may also encompass reference to non-human animals, preferablywarm-blooded animals, more preferably mammals, such as, e.g., non-humanprimates, rodents, canines, felines, equines, ovines, porcines, and thelike.

The terms “sample” or “biological sample” as used herein include anybiological specimen obtained from a subject. Samples may include,without limitation, whole blood, plasma, serum, red blood cells, whiteblood cells (e.g., peripheral blood mononuclear cells), saliva, urine,stool (i.e., faeces), tears, sweat, sebum, nipple aspirate, ductallavage, tumour exudates, synovial fluid, cerebrospinal fluid, lymph,fine needle aspirate, amniotic fluid, any other bodily fluid, celllysates, cellular secretion products, inflammation fluid, semen andvaginal secretions. Preferred samples may include ones comprisingPERLECAN protein in detectable quantities. In preferred embodiments, thesample may be whole blood or a fractional component thereof such as,e.g., plasma, serum, or a cell pellet. Preferably the sample is readilyobtainable by minimally invasive methods, allowing to remove or isolatesaid sample from the subject. Samples may also include tissue samplesand biopsies, tissue homogenates and the like. Preferably, the sampleused to detect PERLECAN levels is blood plasma. Also preferably, thesample used to detect PERLECAN levels is urine. The term “plasma”defines the colorless watery fluid of the blood that contains no cells,but in which the blood cells (erythrocytes, leukocytes, thrombocytes,etc.) are suspended, containing nutrients, sugars, proteins, minerals,enzymes, etc.

A molecule or analyte such as a protein, polypeptide or peptide, or agroup of two or more molecules or analytes such as two or more proteins,polypeptides or peptides, is “measured” in a sample when the presence orabsence and/or quantity of said molecule or analyte or of said group ofmolecules or analytes is detected or determined in the sample,preferably substantially to the exclusion of other molecules andanalytes.

The terms “quantity”, “amount” and “level” are synonymous and generallywell-understood in the art. The terms as used herein may particularlyrefer to an absolute quantification of a molecule or an analyte in asample, or to a relative quantification of a molecule or analyte in asample, i.e., relative to another value such as relative to a referencevalue as taught herein, or to a range of values indicating a base-lineexpression of the biomarker. These values or ranges can be obtained froma single patient or from a group of patients.

An absolute quantity of a molecule or analyte in a sample may beadvantageously expressed as weight or as molar amount, or more commonlyas a concentration, e.g., weight per volume or mol per volume.

A relative quantity of a molecule or analyte in a sample may beadvantageously expressed as an increase or decrease or as afold-increase or fold-decrease relative to said another value, such asrelative to a reference value as taught herein. Performing a relativecomparison between first and second parameters (e.g., first and secondquantities) may but need not require to first determine the absolutevalues of said first and second parameters. For example, a measurementmethod can produce quantifiable readouts (such as, e.g., signalintensities) for said first and second parameters, wherein said readoutsare a function of the value of said parameters, and wherein saidreadouts can be directly compared to produce a relative value for thefirst parameter vs. the second parameter, without the actual need tofirst convert the readouts to absolute values of the respectiveparameters.

As used herein, the term “PERLECAN” corresponds to the protein commonlyknown as PERLECAN (PLC), also known as heparan sulfate proteoglycan 2(HSPG2), sometimes abbreviated as SJA, SJS, SJS1, or PRCAN, i.e. theproteins and polypeptides commonly known under these designations in theart. The terms encompass such proteins and polypeptides of any organismwhere found, and particularly of animals, preferably vertebrates, morepreferably mammals, including humans and non-human mammals, even morepreferably of humans. The terms particularly encompass such proteins andpolypeptides with a native sequence, i.e., ones of which the primarysequence is the same as that of PERLECAN found in or derived fromnature. A skilled person understands that native sequences of PERLECANmay differ between different species due to genetic divergence betweensuch species. Moreover, the native sequences of PERLECAN may differbetween or within different individuals of the same species due tonormal genetic diversity (variation) within a given species. Also, thenative sequences of PERLECAN may differ between or even within differentindividuals of the same species due to post-transcriptional orpost-translational modifications. Accordingly, all PERLECAN sequencesfound in or derived from nature are considered “native”. The termsencompass PERLECAN proteins and polypeptides when forming a part of aliving organism, organ, tissue or cell, when forming a part of abiological sample, as well as when at least partly isolated from suchsources. The terms also encompass proteins and polypeptides whenproduced by recombinant or synthetic means.

Exemplary PERLECAN includes, without limitation, human PERLECAN havingprimary amino acid sequence as annotated under NCBI Genbank accessionnumber NP_005520 (sequence version 4), comprising 4391 amino acids asreproduced in FIG. 1 (SEQ ID NO: 1). A skilled person can alsoappreciate that said sequences are of precursor of PERLECAN and mayinclude parts which are processed away from mature PERLECAN. Forexample, in FIG. 1, the C-terminal endorepellin (SEQ ID NO. 2) and LG3(SEQ ID NO. 3) domains are also indicated. The term “PERLECAN” as usedherein encompasses full-length PERLECAN as well as fragments thereof.Preferred examples of such fragments are endorepellin and/or LG3.

In an embodiment the circulating PERLECAN, e.g., secreted formcirculating in the blood plasma, may be detected, as opposed to thecell-bound or cell-confined PERLECAN protein.

The reference herein to PERLECAN may thus also encompass fragments ofPERLECAN. Hence, the reference herein to measuring PERLECAN, or tomeasuring the quantity of PERLECAN, may encompass measuring the PERLECANprotein or polypeptide, such as, e.g., measuring the mature and/or theprocessed soluble/secreted form (e.g. plasma circulating form) ofPERLECAN and/or measuring one or more fragments thereof, such asendorepellin and/or LG3. For example, PERLECAN and/or one or morefragments thereof may be measured collectively, such that the measuredquantity corresponds to the sum amounts of the collectively measuredspecies, by for example using a binding molecule that binds at theC-terminal end of PERLECAN. In another example, PERLECAN and/or one ormore fragments thereof such as Endorepellin and/or LG3 may be measuredeach individually. Preferably, said fragment of PERLECAN is a plasmacirculating form of PERLECAN. The expression “plasma circulating form ofPERLECAN” or shortly “circulating form” encompasses all PERLECANproteins or fragments thereof that circulate in the plasma, i.e., arenot cell- or membrane-bound. Without wanting to be bound by any theory,such circulating forms can be derived from the full-length PERLECANprotein through natural processing, or can be resulting from knowndegradation processes occurring in said sample. In certain situations,the circulating form can also be the full-length PERLECAN protein, whichis found to be circulating in the plasma. Said “circulating form” canthus be any PERLECAN protein or any processed soluble form of PERLECANor fragments of either one, that is circulating in the sample, i.e.which is not bound to a cell- or membrane fraction of said sample.Exemplary fragments may be the processed Endorepellin or LG-3 peptides.It has for example been reported that the LG3 domain is cleaved from theEndorepellin domain by Cathepsin-L, during the process of apoptosis ofendothelial cells (Cailhier et al., 2008, JBC Vol. 283(40):27220-27229).Endorepellin and especially its LG3 peptide is known to be anangiogenesis inhibitor (Mongiat et al., 2003, JBC Vol.278(6):4238-4249). PERLECAN has for example been shown to be situated inthe basement membrane of e.g. human placenta and decidua, where itappears to be important in maintaining blood vessel and villousintegrity or modelling thereof (Chen et al., 2008, Placenta 29:309-316).

In the literature however, PERLECAN levels in the blood of humansubjects has not been used or suggested for having a diagnostic valuetowards renal dysfunction. Although there are two publications thatdetected a PERLECAN fragment in urine of end-stage renal patients (Odaet al., Clinica Chimica Alta 255(1996):119-132), or chronic allograftnephropathy caused by acute rejection of a renal inplant (O'Riordan etal., 2008 Proteomics Clin. Appl. 2:1025-1035), it could not beanticipated that PERLECAN levels in blood samples could be indicative ofrenal failure. The publication actually reports on a new method forpurifying low molecular weight proteins from urine of patients withend-stage renal disease. No indication is given that said peptide couldbe a diagnostic marker.

The peptides detected in the samples of the subjects according to themethods and use of the present invention are situated in the C-terminalpart of PERLECAN, more precisely in the endorepellin domain, even moreprecisely in the LG-3 domain (cf. FIG. 1). Without wanting to be boundby any theory, an elevated PERLECAN level measured in the samples canthus be linked to an increase in general PERLECAN protein expressionand/or in an increased proteolysis or processing of the PERLECANprotein, which is generally situated in the extracellular matrix,wherein the cleaved forms (e.g. endorepellin and/or LG-3) are releasedfrom the full-length molecule.

As used herein, the terms “pro-B-type natriuretic peptide” (alsoabbreviated as “proBNP”) and “amino terminal pro-B-type natriureticpeptide” (also abbreviated as “NTproBNP”) and “B-type natriureticpeptide” (also abbreviated as “BNP”) refer to peptides commonly knownunder these designations in the art. As further explanation and withoutlimitation, in vivo proBNP, NTproBNP and BNP derive from natriureticpeptide precursor B preproprotein (preproBNP). In particular, proBNPpeptide corresponds to the portion of preproBNP after removal of theN-terminal secretion signal (leader) sequence from preproBNP. NTproBNPcorresponds to the N-terminal portion and BNP corresponds to theC-terminal portion of the proBNP peptide subsequent to cleavage of thelatter C-terminally adjacent to amino acid 76 of proBNP.

The term “Cystatin C”, also known as ARMD11; MGC117328, Cystatin-3(CST3), refers to peptides commonly known under these designations inthe art, as exemplarily annotated under Genbank accession numberNP_000090 (sequence version 1).

As used herein, “neutrophil gelatinase-associated lipocalin” or “NGAL”,also known as oncogenic lipocalin 24P3, uterocalin or lipocalin 2(LCN2), refers to peptides commonly known under these designations inthe art, as exemplarily annotated under Genbank accession numberNP_005555 (sequence version 2).

The term “C-reactive protein”, also known as CRP or PTX1, refers topeptides commonly known under these designations in the art, asexemplarily annotated under Genbank accession number NP_000558 (sequenceversion 2).

The term “beta-trace protein”, also known as inter alia prostaglandin-H2D-isomerase, prostaglandin-D2 synthase, cerebrin-28 and PTGDS, refers topeptides commonly known under these designations in the art, asexemplarily annotated under Genbank accession number NP_000945 (sequenceversion 3).

The term “kidney injury molecule 1” or KIM-1 refers to peptides commonlyknown under these designations in the art, as exemplarily disclosed inIchimura et al. 2004 (Am J Physiol Renal Physiol 286(3): F552-63) andIchimura et al. 1998 (J Biol Chem 273: 4135-4142).

The term “interleukin-18” refers to peptides commonly known under thisdesignation in the art, as exemplarily annotated under Genbank accessionnumber NP_001553 (sequence version 1).

Unless otherwise apparent from the context, reference herein to anyprotein, polypeptide or peptide encompasses such from any organism wherefound, and particularly preferably from animals, preferably vertebrates,more preferably mammals, including humans and non-human mammals, evenmore preferably from humans.

Further, unless otherwise apparent from the context, reference herein toany protein, polypeptide or peptide and fragments thereof may generallyalso encompass modified forms of said protein, polypeptide or peptideand fragments such as bearing post-expression modifications including,for example, phosphorylation, glycosylation, lipidation, methylation,cysteinylation, sulphonation, glutathionylation, acetylation, oxidationof methionine to methionine sulphoxide or methionine sulphone, and thelike.

In an embodiment, PERLECAN and fragments thereof, or other biomarkers asemployed herein and fragments thereof, may be human, i.e., their primarysequence may be the same as a corresponding primary sequence of orpresent in a naturally occurring human peptides, polypeptides orproteins. Hence, the qualifier “human” in this connection relates to theprimary sequence of the respective proteins, polypeptides, peptides orfragments, rather than to their origin or source. For example, suchproteins, polypeptides, peptides or fragments may be present in orisolated from samples of human subjects or may be obtained by othermeans (e.g., by recombinant expression, cell-free translation ornon-biological peptide synthesis).

The term “fragment” of a protein, polypeptide or peptide generallyrefers to N-terminally and/or C-terminally deleted or truncated forms ofsaid protein, polypeptide or peptide. The term encompasses fragmentsarising by any mechanism, such as, without limitation, by alternativetranslation, exo- and/or endo-proteolysis and/or degradation of saidprotein or polypeptide, such as, for example, in vivo or in vitro, suchas, for example, by physical, chemical and/or enzymatic proteolysis.Without limitation, a fragment of a protein, polypeptide or peptide mayrepresent at least about 5%, or at least about 10%, e.g., ≧20%, ≧30% or≧40%, such as ≧50%, e.g., ≧60%, ≧70% or ≧80%, or even ≧90% or ≧95% ofthe amino acid sequence of said protein, polypeptide or peptide.

For example, a fragment may include a sequence of ≧5 consecutive aminoacids, or ≧10 consecutive amino acids, or ≧20 consecutive amino acids,or ≧30 consecutive amino acids, e.g., ≧40 consecutive amino acids, suchas for example ≧50 consecutive amino acids, e.g., ≧60, ≧70, ≧80, ≧90,≧100, ≧200, ≧300, ≧400, ≧500 or ≧600 consecutive amino acids of thecorresponding full length protein.

In an embodiment, a fragment may be N-terminally and/or C-terminallytruncated by between 1 and about 20 amino acids, such as, e.g., bybetween 1 and about 15 amino acids, or by between 1 and about 10 aminoacids, or by between 1 and about 5 amino acids, compared to thecorresponding mature, full-length protein or its soluble or plasmacirculating form. By means of example, proBNP, NTproBNP and BNPfragments useful as biomarkers are disclosed in WO 2004/094460.

In an embodiment, fragments of a given protein, polypeptide or peptidemay be achieved by in vitro proteolysis of said protein, polypeptide orpeptide to obtain advantageously detectable peptide(s) from a sample.For example, such proteolysis may be effected by suitable physical,chemical and/or enzymatic agents, e.g., proteinases, preferablyendoproteinases, i.e., protease cleaving internally within a protein,polypeptide or peptide chain. A non-limiting list of suitableendoproteinases includes serine proteinases (EC 3.4.21), threonineproteinases (EC 3.4.25), cysteine proteinases (EC 3.4.22), aspartic acidproteinases (EC 3.4.23), metalloproteinases (EC 3.4.24) and glutamicacid proteinases. Exemplary non-limiting endoproteinases includetrypsin, chymotrypsin, elastase, Lysobacter enzymogenes endoproteinaseLys-C, Staphylococcus aureus endoproteinase Glu-C (endopeptidase V8) orClostridium histolyticum endoproteinase Arg-C (clostripain). Furtherknown or yet to be identified enzymes may be used; a skilled person canchoose suitable protease(s) on the basis of their cleavage specificityand frequency to achieve desired peptide forms. Preferably, theproteolysis may be effected by endopeptidases of the trypsin type (EC3.4.21.4), preferably trypsin, such as, without limitation, preparationsof trypsin from bovine pancreas, human pancreas, porcine pancreas,recombinant trypsin, Lys-acetylated trypsin, trypsin in solution,trypsin immobilised to a solid support, etc. Trypsin is particularlyuseful, inter alia due to high specificity and efficiency of cleavage.The invention also contemplates the use of any trypsin-like protease,i.e., with a similar specificity to that of trypsin. Otherwise, chemicalreagents may be used for proteolysis. For example, CNBr can cleave atMet; BNPS-skatole can cleave at Trp. The conditions for treatment, e.g.,protein concentration, enzyme or chemical reagent concentration, pH,buffer, temperature, time, can be determined by the skilled persondepending on the enzyme or chemical reagent employed.

Also provided is thus an isolated fragment of PERLECAN as defined hereabove. Such fragments may give useful information about the presence andquantity of PERLECAN in biological samples, whereby the detection ofsaid fragments is of interest. Hence, the herein disclosed fragments ofPERLECAN are useful biomarkers. A preferred PERLECAN fragment maycomprise, consist essentially of or consist of the sequence as set forthin SEQ ID NO: 2.

The term “isolated” with reference to a particular component (such asfor instance, a protein, polypeptide, peptide or fragment thereof)generally denotes that such component exists in separation from—forexample, has been separated from or prepared in separation from—one ormore other components of its natural environment. For instance, anisolated human or animal protein, polypeptide, peptide or fragmentexists in separation from a human or animal body where it occursnaturally.

The term “isolated” as used herein may preferably also encompass thequalifier “purified”. As used herein, the term “purified” with referenceto protein(s), polypeptide(s), peptide(s) and/or fragment(s) thereofdoes not require absolute purity. Instead, it denotes that suchprotein(s), polypeptide(s), peptide(s) and/or fragment(s) is (are) in adiscrete environment in which their abundance (conveniently expressed interms of mass or weight or concentration) relative to other proteins isgreater than in a biological sample. A discrete environment denotes asingle medium, such as for example a single solution, gel, precipitate,lyophilisate, etc. Purified peptides, polypeptides or fragments may beobtained by known methods including, for example, laboratory orrecombinant synthesis, chromatography, preparative electrophoresis,centrifugation, precipitation, affinity purification, etc.

Purified protein(s), polypeptide(s), peptide(s) and/or fragment(s) maypreferably constitute by weight ≧10%, more preferably ≧50%, such as≧60%, yet more preferably ≧70%, such as ≧80%, and still more preferably≧90%, such as ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or even 100%, of the proteincontent of the discrete environment. Protein content may be determined,e.g., by the Lowry method (Lowry et al. 1951. J Biol Chem 193: 265),optionally as described by Hartree 1972 (Anal Biochem 48: 422-427).Also, purity of peptides or polypeptides may be determined by SDS-PAGEunder reducing or non-reducing conditions using Coomassie blue or,preferably, silver stain.

Further disclosed are isolated PERLECAN or fragments thereof as taughtherein comprising a detectable label. This facilitates ready detectionof such fragments. The term “label” as used throughout thisspecification refers to any atom, molecule, moiety or biomolecule thatcan be used to provide a detectable and preferably quantifiable read-outor property, and that can be attached to or made part of an entity ofinterest, such as a peptide or polypeptide or a specific-binding agent.Labels may be suitably detectable by mass spectrometric, spectroscopic,optical, colorimetric, magnetic, photochemical, biochemical,immunochemical or chemical means. Labels include without limitationdyes; radiolabels such as ³²P, ³³P, ³⁵S, ¹²⁵I, ¹³¹I; electron-densereagents; enzymes (e.g., horse-radish phosphatise or alkalinephosphatise as commonly used in immunoassays); binding moieties such asbiotin-streptavidin; haptens such as digoxigenin; luminogenic,phosphorescent or fluorogenic moieties; mass tags; and fluorescent dyesalone or in combination with moieties that can suppress or shiftemission spectra by fluorescence resonance energy transfer (FRET).

For example, the label may be a mass-altering label. Preferably, amass-altering label may involve the presence of a distinct stableisotope in one or more amino acids of the peptide vis-à-vis itscorresponding non-labelled peptide. Mass-labelled peptides areparticularly useful as positive controls, standards and calibrators inmass spectrometry applications. In particular, peptides including one ormore distinct isotopes are chemically alike, separatechromatographically and electrophoretically in the same manner and alsoionise and fragment in the same way. However, in a suitable massanalyser such peptides and optionally select fragmentation ions thereofwill display distinguishable m/z ratios and can thus be discriminated.Examples of pairs of distinguishable stable isotopes include H and D,¹²C and ¹³C, ¹⁴N and ¹⁵N or ¹⁶O and ¹⁸O. Usually, peptides and proteinsof biological samples analysed in the present invention maysubstantially only contain common isotopes having high prevalence innature, such as for example H, ¹²C, ¹⁴N and ¹⁶O. In such case, themass-labelled peptide may be labelled with one or more uncommon isotopeshaving low prevalence in nature, such as for instance D, ¹³C, ¹⁵N and/or¹⁸O. It is also conceivable that in cases where the peptides or proteinsof a biological sample would include one or more uncommon isotopes, themass-labelled peptide may comprise the respective common isotope(s).

Isotopically-labelled synthetic peptides may be obtained inter alia bysynthesising or recombinantly producing such peptides using one or moreisotopically-labelled amino acid substrates, or by chemically orenzymatically modifying unlabelled peptides to introduce thereto one ormore distinct isotopes. By means of example and not limitation,D-labelled peptides may be synthesised or recombinantly produced in thepresence of commercially available deuterated L-methionineCH₃—S—CD₂CD₂-CH(NH₂)—COOH or deuterated arginineH₂NC(═NH)—NH—(CD₂)₃-CD(NH₂)—COOH. It shall be appreciated that any aminoacid of which deuterated or ¹⁵N- or ¹³C-containing forms exist may beconsidered for synthesis or recombinant production of labelled peptides.In another non-limiting example, a peptide may be treated with trypsinin H₂ ¹⁶O or H₂ ¹⁸O, leading to incorporation of two oxygens (¹⁶O or¹⁸O, respectively) at the COOH-termini of said peptide (e.g., US2006/105415).

Accordingly, also contemplated is the use of PERLECAN and isolatedfragments thereof as taught herein, optionally comprising a detectablelabel, as (positive) controls, standards or calibrators in qualitativeor quantitative detection assays (measurement methods) of PERLECAN, andparticularly in such methods for predicting, diagnosing, prognosticatingand/or monitoring the diseases or conditions as taught herein insubjects. The proteins, polypeptides or peptides may be supplied in anyform, inter alia as precipitate, vacuum-dried, lyophilisate, in solutionas liquid or frozen, or covalently or non-covalently immobilised onsolid phase, such as for example, on solid chromatographic matrix or onglass or plastic or other suitable surfaces (e.g., as a part of peptidearrays and microarrays). The peptides may be readily prepared, forexample, isolated from natural sources, or prepared recombinantly orsynthetically.

Further disclosed are binding agents capable of specifically binding toany one or more of the isolated fragments of PERLECAN as taught herein.Also disclosed are binding agents capable of specifically binding toonly one of isolated fragments of PERLECAN as taught herein. Bindingagents as intended throughout this specification may include inter aliaan antibody, aptamer, photoaptamer, protein, peptide, peptidomimetic ora small molecule.

A binding agent may be capable of binding both the plasma circulatingform and the cell-bound or retained from of PERLECAN. Preferably, abinding agent may be capable of specifically binding or detecting theplasma circulating form of PERLECAN.

The term “specifically bind” as used throughout this specification meansthat an agent (denoted herein also as “specific-binding agent”) binds toone or more desired molecules or analytes, such as to one or moreproteins, polypeptides or peptides of interest or fragments thereofsubstantially to the exclusion of other molecules which are random orunrelated, and optionally substantially to the exclusion of othermolecules that are structurally related. The term “specifically bind”does not necessarily require that an agent binds exclusively to itsintended target(s). For example, an agent may be said to specificallybind to protein(s) polypeptide(s), peptide(s) and/or fragment(s) thereofof interest if its affinity for such intended target(s) under theconditions of binding is at least about 2-fold greater, preferably atleast about 5-fold greater, more preferably at least about 10-foldgreater, yet more preferably at least about 25-fold greater, still morepreferably at least about 50-fold greater, and even more preferably atleast about 100-fold or more greater, than its affinity for a non-targetmolecule.

Preferably, the agent may bind to its intended target(s) with affinityconstant (K_(A)) of such binding K_(A)≧1×10⁶ M⁻¹, more preferablyK_(A)≧1×10⁷ M⁻¹, yet more preferably K_(A)≧1×10⁸ M⁻¹, even morepreferably K_(A)≧1×10⁹ M⁻¹, and still more preferably K_(A)≧1×10¹⁰ M⁻¹or K_(A)≧1×10¹¹ M⁻¹, wherein K_(A)=[SBA_T]/[SBA][T], SBA denotes thespecific-binding agent, T denotes the intended target. Determination ofK_(A) can be carried out by methods known in the art, such as forexample, using equilibrium dialysis and Scatchard plot analysis.

Specific binding agents as used throughout this specification mayinclude inter alia an antibody, aptamer, photoaptamer, protein, peptide,peptidomimetic or a small molecule.

As used herein, the term “antibody” is used in its broadest sense andgenerally refers to any immunologic binding agent. The term specificallyencompasses intact monoclonal antibodies, polyclonal antibodies,multivalent (e.g., 2-, 3- or more-valent) and/or multi-specificantibodies (e.g., bi- or more-specific antibodies) formed from at leasttwo intact antibodies, and antibody fragments insofar they exhibit thedesired biological activity (particularly, ability to specifically bindan antigen of interest), as well as multivalent and/or multi-specificcomposites of such fragments. The term “antibody” is not only inclusiveof antibodies generated by methods comprising immunisation, but alsoincludes any polypeptide, e.g., a recombinantly expressed polypeptide,which is made to encompass at least one complementarity-determiningregion (CDR) capable of specifically binding to an epitope on an antigenof interest. Hence, the term applies to such molecules regardlesswhether they are produced in vitro or in vivo.

An antibody may be any of IgA, IgD, IgE, IgG and IgM classes, andpreferably IgG class antibody. An antibody may be a polyclonal antibody,e.g., an antiserum or immunoglobulins purified there from (e.g.,affinity-purified). An antibody may be a monoclonal antibody or amixture of monoclonal antibodies. Monoclonal antibodies can target aparticular antigen or a particular epitope within an antigen withgreater selectivity and reproducibility. By means of example and notlimitation, monoclonal antibodies may be made by the hybridoma methodfirst described by Kohler et al. 1975 (Nature 256: 495), or may be madeby recombinant DNA methods (e.g., as in U.S. Pat. No. 4,816,567).Monoclonal antibodies may also be isolated from phage antibody librariesusing techniques as described by Clackson et al. 1991 (Nature 352:624-628) and Marks et al. 1991 (J Mol Biol 222: 581-597), for example.

Antibody binding agents may be antibody fragments. “Antibody fragments”comprise a portion of an intact antibody, comprising the antigen-bindingor variable region thereof. Examples of antibody fragments include Fab,Fab′, F(ab′)2, Fv and scFv fragments; diabodies; linear antibodies;single-chain antibody molecules; and multivalent and/or multispecificantibodies formed from antibody fragment(s), e.g., dibodies, tribodies,and multibodies. The above designations Fab, Fab′, F(ab′)2, Fv, scFvetc. are intended to have their art-established meaning.

The term antibody includes antibodies originating from or comprising oneor more portions derived from any animal species, preferably vertebratespecies, including, e.g., birds and mammals. Without limitation, theantibodies may be chicken, turkey, goose, duck, guinea fowl, quail orpheasant. Also without limitation, the antibodies may be human, murine(e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel(e.g., Camelus bactrianus and Camelus dromaderius), llama (e.g., Lamapaccos, Lama glama or Lama vicugna) or horse.

A skilled person will understand that an antibody can include one ormore amino acid deletions, additions and/or substitutions (e.g.,conservative substitutions), insofar such alterations preserve itsbinding of the respective antigen. An antibody may also include one ormore native or artificial modifications of its constituent amino acidresidues (e.g., glycosylation, etc.).

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art, as are methods to producerecombinant antibodies or fragments thereof (see for example, Harlow andLane, “Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory,New York, 1988; Harlow and Lane, “Using Antibodies: A LaboratoryManual”, Cold Spring Harbour Laboratory, New York, 1999, ISBN0879695447; “Monoclonal Antibodies: A Manual of Techniques”, by Zola,ed., CRC Press 1987, ISBN 0849364760; “Monoclonal Antibodies: APractical Approach”, by Dean & Shepherd, eds., Oxford University Press2000, ISBN 0199637229; Methods in Molecular Biology, vol. 248: “AntibodyEngineering: Methods and Protocols”, Lo, ed., Humana Press 2004, ISBN1588290921).

The term “aptamer” refers to single-stranded or double-strandedoligo-DNA, oligo-RNA or oligo-DNA/RNA or any analogue thereof, that canspecifically bind to a target molecule such as a peptide.Advantageously, aptamers can display fairly high specificity andaffinity (e.g., K_(A) in the order 1×10⁹ M⁻¹) for their targets. Aptamerproduction is described inter alia in U.S. Pat. No. 5,270,163; Ellington& Szostak 1990 (Nature 346: 818-822); Tuerk & Gold 1990 (Science 249:505-510); or “The Aptamer Handbook: Functional Oligonucleotides andTheir Applications”, by Klussmann, ed., Wiley-VCH 2006, ISBN 3527310592,incorporated by reference herein. The term “photoaptamer” refers to anaptamer that contains one or more photoreactive functional groups thatcan covalently bind to or crosslink with a target molecule. The term“peptidomimetic” refers to a non-peptide agent that is a topologicalanalogue of a corresponding peptide. Methods of rationally designingpeptidomimetics of peptides are known in the art. For example, therational design of three peptidomimetics based on the sulphated 8-merpeptide CCK26-33, and of two peptidomimetics based on the 11-mer peptideSubstance P, and related peptidomimetic design principles, are describedin Norwell 1995 (Trends Biotechnol 13: 132-134).

The term “small molecule” refers to compounds, preferably organiccompounds, with a size comparable to those organic molecules generallyused in pharmaceuticals. The term excludes biological macromolecules(e.g., proteins, nucleic acids, etc.). Preferred small organic moleculesrange in size up to about 5000 Da, e.g., up to about 4000, preferably upto 3000 Da, more preferably up to 2000 Da, even more preferably up toabout 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500Da.

Hence, also disclosed are methods for immunising animals, e.g.,non-human animals such as laboratory or farm, animals using (i.e., usingas the immunising antigen) the herein taught fragments of PERLECAN,optionally attached to a presenting carrier. Immunisation andpreparation of antibody reagents from immune sera is well-known per seand described in documents referred to elsewhere in this specification.The animals to be immunised may include any animal species, preferablywarm-blooded species, more preferably vertebrate species, including,e.g., birds and mammals. Without limitation, the antibodies may bechicken, turkey, goose, duck, guinea fowl, quail or pheasant. Alsowithout limitation, the antibodies may be human, murine (e.g., mouse,rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel, llama orhorse. The term “presenting carrier” or “carrier” generally denotes animmunogenic molecule which, when bound to a second molecule, augmentsimmune responses to the latter, usually through the provision ofadditional T cell epitopes. The presenting carrier may be a(poly)peptidic structure or a non-peptidic structure, such as inter aliaglycans, polyethylene glycols, peptide mimetics, synthetic polymers,etc. Exemplary non-limiting carriers include human Hepatitis B viruscore protein, multiple C3d domains, tetanus toxin fragment C or yeast Typarticles.

Immune sera obtained or obtainable by immunisation as taught herein maybe particularly useful for generating antibody reagents thatspecifically bind to one or more of the herein disclosed fragments ofPERLECAN.

Further disclosed are methods for selecting specific-binding agentswhich bind (a) one or more of the PERLECAN fragments taught herein,substantially to the exclusion of (b) PERLECAN and/or other fragmentsthereof. Conveniently, such methods may be based on subtracting orremoving binding agents which cross-react or cross-bind the non-desiredPERLECAN molecules under (b). Such subtraction may be readily performedas known in the art by a variety of affinity separation methods, such asaffinity chromatography, affinity solid phase extraction, affinitymagnetic extraction, etc.

Any existing, available or conventional separation, detection andquantification methods can be used herein to measure the presence orabsence (e.g., readout being present vs. absent; or detectable amountvs. undetectable amount) and/or quantity (e.g., readout being anabsolute or relative quantity, such as, for example, absolute orrelative concentration) of PERLECAN and/or fragments thereof andoptionally of the one or more other biomarkers or fragments thereof insamples (any molecules or analytes of interest to be so-measured insamples, including PERLECAN and fragments thereof, may be herein belowreferred to collectively as biomarkers).

For example, such methods may include immunoassay methods, massspectrometry analysis methods, or chromatography methods, orcombinations thereof.

The term “immunoassay” generally refers to methods known as such fordetecting one or more molecules or analytes of interest in a sample,wherein specificity of an immunoassay for the molecule(s) or analyte(s)of interest is conferred by specific binding between a specific-bindingagent, commonly an antibody, and the molecule(s) or analyte(s) ofinterest. Immunoassay technologies include without limitation directELISA (enzyme-linked immunosorbent assay), indirect ELISA, sandwichELISA, competitive ELISA, multiplex ELISA, radioimmunoassay (RIA),ELISPOT technologies, and other similar techniques known in the art.Principles of these immunoassay methods are known in the art, forexample John R. Crowther, “The ELISA Guidebook”, 1st ed., Humana Press2000, ISBN 0896037282.

By means of further explanation and not limitation, direct ELISA employsa labelled primary antibody to bind to and thereby quantify targetantigen in a sample immobilised on a solid support such as a microwellplate. Indirect ELISA uses a non-labelled primary antibody which bindsto the target antigen and a secondary labelled antibody that recognisesand allows to quantify the antigen-bound primary antibody. In sandwichELISA the target antigen is captured from a sample using an immobilised‘capture’ antibody which binds to one antigenic site within the antigen,and subsequent to removal of non-bound analytes the so-captured antigenis detected using a ‘detection’ antibody which binds to anotherantigenic site within said antigen, where the detection antibody may bedirectly labelled or indirectly detectable as above. Competitive ELISAuses a labelled ‘competitor’ that may either be the primary antibody orthe target antigen. In an example, non-labelled immobilised primaryantibody is incubated with a sample, this reaction is allowed to reachequilibrium, and then labelled target antigen is added. The latter willbind to the primary antibody wherever its binding sites are not yetoccupied by non-labelled target antigen from the sample. Thus, thedetected amount of bound labelled antigen inversely correlates with theamount of non-labelled antigen in the sample. Multiplex ELISA allowssimultaneous detection of two or more analytes within a singlecompartment (e.g., microplate well) usually at a plurality of arrayaddresses (see, for example, Nielsen & Geierstanger 2004. J ImmunolMethods 290: 107-20 and Ling et al. 2007. Expert Rev Mol Diagn 7: 87-98for further guidance). As appreciated, labelling in ELISA technologiesis usually by enzyme (such as, e.g., horse-radish peroxidase)conjugation and the end-point is typically colorimetric,chemiluminescent or fluorescent, magnetic, piezo electric, pyroelectricand other.

Radioimmunoassay (RIA) is a competition-based technique and involvesmixing known said antigen, then adding non-labelled or ‘cold’ antigenfrom a sample and measuring the amount of labelled antigen displaced(see, e.g., “An Introduction to Radioimmunoassay and RelatedTechniques”, by Chard T, ed., Elsevier Science 1995, ISBN 0444821198 forguidance).

Generally, any mass spectrometric (MS) techniques that can obtainprecise information on the mass of peptides, and preferably also onfragmentation and/or (partial) amino acid sequence of selected peptides(e.g., in tandem mass spectrometry, MS/MS; or in post source decay, TOFMS), are useful herein. Suitable peptide MS and MS/MS techniques andsystems are well-known per se (see, e.g., Methods in Molecular Biology,vol. 146: “Mass Spectrometry of Proteins and Peptides”, by Chapman, ed.,Humana Press 2000, ISBN 089603609x; Biemann 1990. Methods Enzymol 193:455-79; or Methods in Enzymology, vol. 402: “Biological MassSpectrometry”, by Burlingame, ed., Academic Press 2005, ISBN9780121828073) and may be used herein. MS arrangements, instruments andsystems suitable for biomarker peptide analysis may include, withoutlimitation, matrix-assisted laser desorption/ionisation time-of-flight(MALDI-TOF) MS; MALDI-TOF post-source-decay (PSD); MALDI-TOF/TOF;surface-enhanced laser desorption/ionization time-of-flight massspectrometry (SELDI-TOF) MS; electrospray ionization mass spectrometry(ESI-MS); ESI-MS/MS; ESI-MS/(MS)^(n) (n is an integer greater thanzero); ESI 3D or linear (2D) ion trap MS; ESI triple quadrupole MS; ESIquadrupole orthogonal TOF (Q-TOF); ESI Fourier transform MS systems;desorption/ionization on silicon (DIOS); secondary ion mass spectrometry(SIMS); atmospheric pressure chemical ionization mass spectrometry(APCI-MS); APCI-MS/MS; APCI-(MS)^(n); atmospheric pressurephotoionization mass spectrometry (APPI-MS); APPI-MS/MS; andAPPI-(MS)^(n). Peptide ion fragmentation in tandem MS (MS/MS)arrangements may be achieved using manners established in the art, suchas, e.g., collision induced dissociation (CID). Detection andquantification of biomarkers by mass spectrometry may involve multiplereaction monitoring (MRM), such as described among others by Kuhn et al.2004 (Proteomics 4: 1175-86). MS peptide analysis methods may beadvantageously combined with upstream peptide or protein separation orfractionation methods, such as for example with the chromatographic andother methods described herein below.

Chromatography can also be used for measuring biomarkers. As usedherein, the term “chromatography” encompasses methods for separatingchemical substances, referred to as such and vastly available in theart. In a preferred approach, chromatography refers to a process inwhich a mixture of chemical substances (analytes) carried by a movingstream of liquid or gas (“mobile phase”) is separated into components asa result of differential distribution of the analytes, as they flowaround or over a stationary liquid or solid phase (“stationary phase”),between said mobile phase and said stationary phase. The stationaryphase may be usually a finely divided solid, a sheet of filter material,or a thin film of a liquid on the surface of a solid, or the like.Chromatography is also widely applicable for the separation of chemicalcompounds of biological origin, such as, e.g., amino acids, proteins,fragments of proteins or peptides, etc.

Chromatography as used herein may be preferably columnar (i.e., whereinthe stationary phase is deposited or packed in a column), preferablyliquid chromatography, and yet more preferably HPLC. While particularsof chromatography are well known in the art, for further guidance see,e.g., Meyer M., 1998, ISBN: 047198373X, and “Practical HPLC Methodologyand Applications”, Bidlingmeyer, B. A., John Wiley & Sons Inc., 1993.Exemplary types of chromatography include, without limitation,high-performance liquid chromatography (HPLC), normal phase HPLC(NP-HPLC), reversed phase HPLC (RP-HPLC), ion exchange chromatography(IEC), such as cation or anion exchange chromatography, hydrophilicinteraction chromatography (HILIC), hydrophobic interactionchromatography (HIC), size exclusion chromatography (SEC) including gelfiltration chromatography or gel permeation chromatography,chromatofocusing, affinity chromatography such as immuno-affinity,immobilised metal affinity chromatography, and the like.

Chromatography, including single-, two- or more-dimensionalchromatography, may be used as a peptide fractionation method inconjunction with a further peptide analysis method, such as for example,with a downstream mass spectrometry analysis as described elsewhere inthis specification.

Further peptide or polypeptide separation, identification orquantification methods may be used, optionally in conjunction with anyof the above described analysis methods, for measuring biomarkers in thepresent disclosure. Such methods include, without limitation, chemicalextraction partitioning, isoelectric focusing (IEF) including capillaryisoelectric focusing (CIEF), capillary isotachophoresis (CITP),capillary electrochromatography (CEC), and the like, one-dimensionalpolyacrylamide gel electrophoresis (PAGE), two-dimensionalpolyacrylamide gel electrophoresis (2D-PAGE), capillary gelelectrophoresis (CGE), capillary zone electrophoresis (CZE), micellarelectrokinetic chromatography (MEKC), free flow electrophoresis (FFE),etc.

The various aspects and embodiments taught herein may further rely oncomparing the quantity of PERLECAN, as defined herein, measured insamples with reference values of the quantity of PERLECAN, wherein saidreference values represent known predictions, diagnoses and/or prognosesof diseases or conditions as taught herein.

For example, distinct reference values may represent the prediction of arisk (e.g., an abnormally elevated risk) of having a given disease orcondition as taught herein vs. the prediction of no or normal risk ofhaving said disease or condition. In another example, distinct referencevalues may represent predictions of differing degrees of risk of havingsuch disease or condition.

In a further example, distinct reference values can represent thediagnosis of a given disease or condition as taught herein vs. thediagnosis of no such disease or condition (such as, e.g., the diagnosisof healthy, or recovered from said disease or condition, etc.). Inanother example, distinct reference values may represent the diagnosisof such disease or condition of varying severity.

In yet another example, distinct reference values may represent a goodprognosis for a given disease or condition as taught herein vs. a poorprognosis for said disease or condition. In a further example, distinctreference values may represent varyingly favourable or unfavourableprognoses for such disease or condition.

Such comparison may generally include any means to determine thepresence or absence of at least one difference and optionally of thesize of such different between values or profiles being compared. Acomparison may include a visual inspection, an arithmetical orstatistical comparison of measurements. Such statistical comparisonsinclude, but are not limited to, applying a rule. If the values orbiomarker profiles comprise at least one standard, the comparison todetermine a difference in said values or biomarker profiles may alsoinclude measurements of these standards, such that measurements of thebiomarker are correlated to measurements of the internal standards.

Reference values for the quantity of PERLECAN may be establishedaccording to known procedures previously employed for other biomarkers.

For example, a reference value of the quantity of PERLECAN for aparticular prediction, diagnosis and/or prognosis of given disease orcondition as taught herein may be established by determining thequantity of PERLECAN in sample(s) from one individual or from apopulation of individuals characterised by said particular prediction,diagnosis and/or prognosis of said disease or condition (i.e., for whomsaid prediction, diagnosis and/or prognosis of renal dysfunction holdstrue). Such population may comprise without limitation 2, 10, 100, oreven several hundreds or more individuals.

Hence, by means of an illustrative example, reference values of thequantity of PERLECAN for the diagnoses of a given disease or conditionas taught herein vs. no such disease or condition may be established bydetermining the quantity of PERLECAN in sample(s) from one individual orfrom a population of individuals diagnosed (e.g., based on otheradequately conclusive means, such as, for example, clinical signs andsymptoms, imaging, ECG, etc.) as, respectively, having or not havingsaid disease or condition.

In an embodiment, reference value(s) as intended herein may conveyabsolute quantities of PERLECAN. In another embodiment, the quantity ofPERLECAN in a sample from a tested subject may be determined directlyrelative to the reference value (e.g., in terms of increase or decrease,or fold-increase or fold-decrease). Advantageously, this may allow tocompare the quantity of PERLECAN in the sample from the subject with thereference value (in other words to measure the relative quantity ofPERLECAN in the sample from the subject vis-à-vis the reference value)without the need to first determine the respective absolute quantitiesof PERLECAN.

The expression level or presence of a biomarker in a sample of a patientmay sometimes fluctuate, i.e. increase or decrease significantly withoutchange (appearance of, worsening or improving of) symptoms. In such anevent, the marker change precedes the change in symptoms and becomes amore sensitive measure than symptom change. Therapeutic intervention canbe initiated earlier and be more effective than waiting fordeteriorating symptoms. Early intervention at a more benign status maybe carried out safely at home, which is a major improvement fromtreating seriously deteriorated patients in the emergency room.

Measuring the PERLECAN level of the same patient at different timepoints can in such a case thus enable the continuous monitoring of thestatus of the patient and can lead to prediction of worsening orimprovement of the patient's condition with regard to a given disease orcondition as taught herein. A home or clinical test kit or device asindicated herein can be used for this continuous monitoring. One or morereference values or ranges of PERLECAN levels linked to a certaindisease state (e.g. renal dysfunction or no renal dysfunction) for sucha test can e.g. be determined beforehand or during the monitoringprocess over a certain period of time in said subject. Alternatively,these reference values or ranges can be established through data sets ofseveral patients with highly similar disease phenotypes, e.g. fromhealthy subjects or subjects not having the disease or condition ofinterest. A sudden deviation of the PERLECAN levels from said referencevalue or range can predict the worsening of the condition of the patient(e.g. at home or in the clinic) before the (often severe) symptomsactually can be felt or observed.

Also disclosed is thus a method or algorithm for determining asignificant change in the level of the PERLECAN marker in a certainpatient, which is indicative for change (worsening or improving) inclinical status. In addition, the invention allows establishing thediagnosis that the subject is recovering or has recovered from a givendisease or condition as taught herein.

In an embodiment the present methods may include a step of establishingsuch reference value(s). In an embodiment, the present kits and devicesmay include means for establishing a reference value of the quantity ofPERLECAN for a particular prediction, diagnosis and/or prognosis of agiven disease or condition as taught herein. Such means may for examplecomprise one or more samples (e.g., separate or pooled samples) from oneor more individuals characterised by said particular prediction,diagnosis and/or prognosis of said disease or condition.

The various aspects and embodiments taught herein may further entailfinding a deviation or no deviation between the quantity of PERLECANmeasured in a sample from a subject and a given reference value.

A “deviation” of a first value from a second value may generallyencompass any direction (e.g., increase: first value>second value; ordecrease: first value<second value) and any extent of alteration.

For example, a deviation may encompass a decrease in a first value by,without limitation, at least about 10% (about 0.9-fold or less), or byat least about 20% (about 0.8-fold or less), or by at least about 30%(about 0.7-fold or less), or by at least about 40% (about 0.6-fold orless), or by at least about 50% (about 0.5-fold or less), or by at leastabout 60% (about 0.4-fold or less), or by at least about 70% (about0.3-fold or less), or by at least about 80% (about 0.2-fold or less), orby at least about 90% (about 0.1-fold or less), relative to a secondvalue with which a comparison is being made.

For example, a deviation may encompass an increase of a first value by,without limitation, at least about 10% (about 1.1-fold or more), or byat least about 20% (about 1.2-fold or more), or by at least about 30%(about 1.3-fold or more), or by at least about 40% (about 1.4-fold ormore), or by at least about 50% (about 1.5-fold or more), or by at leastabout 60% (about 1.6-fold or more), or by at least about 70% (about1.7-fold or more), or by at least about 80% (about 1.8-fold or more), orby at least about 90% (about 1.9-fold or more), or by at least about100% (about 2-fold or more), or by at least about 150% (about 2.5-foldor more), or by at least about 200% (about 3-fold or more), or by atleast about 500% (about 6-fold or more), or by at least about 700%(about 8-fold or more), or like, relative to a second value with which acomparison is being made.

Preferably, a deviation may refer to a statistically significantobserved alteration. For example, a deviation may refer to an observedalteration which falls outside of error margins of reference values in agiven population (as expressed, for example, by standard deviation orstandard error, or by a predetermined multiple thereof, e.g., ±1×SD or±2×SD, or ±1×SE or ±2×SE). Deviation may also refer to a value fallingoutside of a reference range defined by values in a given population(for example, outside of a range which comprises ≧40%, ≧50%, ≧60%, ≧70%,≧75% or ≧80% or ≧85% or ≧90% or ≧95% or even ≧100% of values in saidpopulation).

In a further embodiment, a deviation may be concluded if an observedalteration is beyond a given threshold or cut-off. Such threshold orcut-off may be selected as generally known in the art to provide for achosen sensitivity and/or specificity of the prediction, diagnosisand/or prognosis methods, e.g., sensitivity and/or specificity of atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 85%, or at least 90%, or at least 95%.

For example, in an embodiment, an elevated quantity of PERLECAN in thesample from the subject—preferably at least about 1.1-fold elevated, orat least about 1.2-fold elevated, more preferably at least about1.3-fold elevated, even more preferably at least about 1.4-foldelevated, yet more preferably at least about 1.5-fold elevated, such asbetween about 1.1-fold and 3-fold elevated or between about 1.5-fold and2-fold elevated—compared to a reference value representing theprediction or diagnosis of no given disease or condition as taughtherein or representing a good prognosis for said disease or conditionindicates that the subject has or is at risk of having said disease orcondition or indicates a poor prognosis for the disease or condition inthe subject.

When a deviation is found between the quantity of PERLECAN in a samplefrom a subject and a reference value representing a certain prediction,diagnosis and/or prognosis of a given disease or condition as taughtherein, said deviation is indicative of or may be attributed to theconclusion that the prediction, diagnosis and/or prognosis of saiddisease or condition in said subject is different from that representedby the reference value.

When no deviation is found between the quantity of PERLECAN in a samplefrom a subject and a reference value representing a certain prediction,diagnosis and/or prognosis of a given disease or condition as taughtherein, the absence of such deviation is indicative of or may beattributed to the conclusion that the prediction, diagnosis and/orprognosis of said disease or condition in said subject is substantiallythe same as that represented by the reference value.

The above considerations apply analogously to biomarker profiles.

When two or more different biomarkers are determined in a subject, theirrespective presence, absence and/or quantity may be together representedas a biomarker profile, the values for each measured biomarker making apart of said profile. As used herein, the term “profile” includes anyset of data that represents the distinctive features or characteristicsassociated with a condition of interest, such as with a particularprediction, diagnosis and/or prognosis of a given disease or conditionas taught herein. The term generally encompasses inter alia nucleic acidprofiles, such as for example genotypic profiles (sets of genotypic datathat represents the genotype of one or more genes associated with acondition of interest), gene copy number profiles (sets of gene copynumber data that represents the amplification or deletion of one or moregenes associated with a condition of interest), gene expression profiles(sets of gene expression data that represents the mRNA levels of one ormore genes associated with a condition of interest), DNA methylationprofiles (sets of methylation data that represents the DNA methylationlevels of one or more genes associated with a condition of interest), aswell as protein, polypeptide or peptide profiles, such as for exampleprotein expression profiles (sets of protein expression data thatrepresents the levels of one or more proteins associated with acondition of interest), protein activation profiles (sets of data thatrepresents the activation or inactivation of one or more proteinsassociated with a condition of interest), protein modification profiles(sets of data that represents the modification of one or more proteinsassociated with a condition of interest), protein cleavage profiles(sets of data that represent the proteolytic cleavage of one or moreproteins associated with a condition of interest), as well as anycombinations thereof.

Biomarker profiles may be created in a number of ways and may be thecombination of measurable biomarkers or aspects of biomarkers usingmethods such as ratios, or other more complex association methods oralgorithms (e.g., rule-based methods). A biomarker profile comprises atleast two measurements, where the measurements can correspond to thesame or different biomarkers. A biomarker profile may also comprise atleast three, four, five, 10, 20, 30 or more measurements. In oneembodiment, a biomarker profile comprises hundreds, or even thousands,of measurements.

Hence, for example, distinct reference profiles may represent theprediction of a risk (e.g., an abnormally elevated risk) of having agiven disease or condition vs. the prediction of no or normal risk ofhaving said disease or condition. In another example, distinct referenceprofiles may represent predictions of differing degrees of risk ofhaving said disease or condition.

In a further example, distinct reference profiles can represent thediagnosis of a given disease or condition as taught herein vs. thediagnosis no such disease or condition (such as, e.g., the diagnosis ofhealthy, recovered from said disease or condition, etc.). In anotherexample, distinct reference profiles may represent the diagnosis of saiddisease or condition of varying severity.

In a yet another example, distinct reference profiles may represent agood prognosis for a disease or condition as taught herein vs. a poorprognosis for said disease or condition. In a further example, distinctreference profiles may represent varyingly favourable or unfavourableprognoses for such disease or condition.

Reference profiles used herein may be established according to knownprocedures previously employed for other biomarkers.

For example, a reference profile of the quantity of PERLECAN and thepresence or absence and/or quantity of one or more other biomarkers fora particular prediction, diagnosis and/or prognosis of a given diseaseor condition as taught herein may be established by determining theprofile in sample(s) from one individual or from a population ofindividuals characterised by said particular prediction, diagnosisand/or prognosis of said disease or condition (i.e., for whom saidprediction, diagnosis and/or prognosis of said disease or conditionholds true). Such population may comprise without limitation ≧2, ≧10,≧100, or even several hundreds or more individuals.

Hence, by means of an illustrative example, reference profiles for thediagnoses of a given disease or condition as taught herein vs. no suchdisease or condition may be established by determining the biomarkerprofiles in sample(s) from one individual or from a population ofindividuals diagnosed as, respectively, having or not having saiddisease or condition.

In an embodiment the present methods may include a step of establishingsuch reference profile(s). In an embodiment, the present kits anddevices may include means for establishing a reference profile for aparticular prediction, diagnosis and/or prognosis of a given disease orcondition as taught herein. Such means may for example comprise one ormore samples (e.g., separate or pooled samples) from one or moreindividuals characterised by said particular prediction, diagnosisand/or prognosis of said disease or condition.

Further, art-known multi-parameter analyses may be employed mutatismutandis to determine deviations between groups of values and profilesgenerated there from (e.g., between sample and reference biomarkerprofiles).

When a deviation is found between the sample profile and a referenceprofile representing a certain prediction, diagnosis and/or prognosis ofa given disease or condition as taught herein, said deviation isindicative of or may be attributed to the conclusion that theprediction, diagnosis and/or prognosis of said disease or condition insaid subject is different from that represented by the referenceprofile.

When no deviation is found between the sample profile and a referenceprofile representing a certain prediction, diagnosis and/or prognosis ofa given disease or condition as taught herein, the absence of suchdeviation is indicative of or may be attributed to the conclusion thatthe prediction, diagnosis and/or prognosis of said disease or conditionin said subject is substantially the same as that represented by thereference profile.

The present invention further provides kits or devices for thediagnosis, prediction, prognosis and/or monitoring of any one disease orcondition as taught herein comprising means for detecting the level ofany one or more biomarkers in a sample of the patient. In a morepreferred embodiment, such a kit or kits of the invention can be used inclinical settings or at home. The kit according to the invention can beused for diagnosing said disease or condition, for monitoring theeffectiveness of treatment of a subject suffering from said disease orcondition with an agent, or for preventive screening of subjects for theoccurrence of said disease or condition in said subject.

In a clinical setting, the kit or device can be in the form of abed-side device or in an emergency team setting, e.g. as part of theequipment of an ambulance or other moving emergency vehicle or teamequipment or as part of a first-aid kit. The diagnostic kit or devicecan assist a medical practitioner, a first aid helper, or nurse todecide whether the patient under observation is developing a disease orcondition as taught herein, after which appropriate action or treatmentcan be performed.

A home-test kit gives the patient a readout which he can communicate toa medicinal practitioner, a first aid helper or to the emergencydepartment of a hospital, after which appropriate action can be taken.Such a home-test device is of particular interest for people havingeither a history of, or are at risk of suffering from any one disease orcondition as taught herein.

Typical kits or devices according to the invention comprise thefollowing elements:

a) a means for obtaining a sample from the subject

b) a means or device for measuring the amount of any one or more markersas taught herein in said sample and visualizing whether the amount ofthe one or more markers in said sample is below or above a certainthreshold level or value, indicating whether the subject is sufferingfrom a given disease or condition as taught herein or not.

In any of the embodiments of the invention, the kits or devices canadditionally comprise c) means for communicating directly with a medicalpractitioner, an emergency department of the hospital or a first aidpost, indicating that a person is suffering from said disease orcondition or not.

The term “threshold level or value” or “reference value” is usedinterchangeably as a synonym and is as defined herein. It can also be arange of base-line (e.g. “dry weight”) values determined in anindividual patient or in a group of patients with highly similar diseaseconditions, taken at about the same time of gestation.

Without wanting to be bound by any theory, the inventors saw that thePERLECAN level is increased in case of renal dysfunction, both at theprotein and mRNA level. In the PE patients tested, the PERLECAN level ishigher than that of healthy subjects.

The threshold value indicated in the present invention is therefore moreto be seen as a value in a reference, i.e. non-PE pregnant subject,taken at about the same stage of gestation and not so much as the valueof the PE-subject before or after pregnancy.

Any of kits as defined herein can be used as a bed-side device for useby the subject himself or by a clinical practitioner.

Non-limiting examples are: systems comprising specific binding moleculesfor said one or more markers attached to a solid phase, e.g. lateralflow strips or dipstick devices and the like well known in the art. Onenon-limiting example to perform a biochemical assay is to use atest-strip and labelled antibodies which combination does not requireany washing of the membrane. The test strip is well known, for example,in the field of pregnancy testing kits where an anti-hCG antibody ispresent on the support, and is carried complexed with hCG by the flow ofurine onto an immobilised second antibody that permits visualisation.Other non-limiting examples of such home test devices, systems or kitscan be found for example in the following U.S. Pat. Nos. 6,107,045,6,974,706, 5,108,889, 6,027,944, 6,482,156, 6,511,814, 5,824,268,5,726,010, 6,001,658 or U.S. patent applications: 2008/0090305 or2003/0109067.

In a preferred embodiment, the invention provides a lateral flow deviceor dipstick. Such dipstick comprises a test strip allowing migration ofa sample by capillary flow from one end of the strip where the sample isapplied to the other end of such strip where presence of an analyte insaid sample is measured. In another embodiment, the invention provides adevice comprising a reagent strip. Such reagent strip comprises one ormore test pads which when wetted with the sample, provide a colourchange in the presence of an analyte and/or indicate the concentrationof the protein in said sample.

In order to obtain a semi-quantitative test strip in which only a signalis formed once the level of any one or more markers in the sample ishigher than a certain predetermined threshold level or value, apredetermined amount of fixed capture antibodies for PERLECAN can bepresent on the test strip. This enables the capture of a certain amountof PERLECAN present in the sample, corresponding to the threshold levelor value as predetermined. The remaining amount of PERLECAN (if any)bound by e.g. a conjugated or labelled binding molecules can then beallowed to migrate to a detection zone which subsequently only producesa signal if the level of said one or more biomarkers in the sample ishigher than the predetermined threshold level or value.

Another possibility to determine whether the amount of any one or moremarkers in the sample is below or above a certain threshold level orvalue, is to use a primary capturing antibody capturing all said one ormore markers protein present in the sample, in combination with alabelled secondary antibody, developing a certain signal or colour whenbound to the solid phase. The intensity of the colour or signal can theneither be compared to a reference colour or signal chart indicating thatwhen the intensity of the signal is above a certain threshold signal,the test is positive. Alternatively, the amount or intensity of thecolour or signal can be measured with an electronic device comprisinge.g. a light absorbance sensor or light emission meter, resulting in anumerical value of signal intensity or colour absorbance formed, whichcan then be displayed to the subject in the form of a negative result ifsaid numerical value is below the threshold value or a positive resultif said numerical value is above the threshold value. This embodiment isof particular relevance in monitoring the level of said one or moremarkers in a patient over a period of time.

The reference value or range can e.g. be determined using the homedevice in a period wherein the subject is free of a given disease orcondition, giving the patient an indication of his base-line level ofany one or more markers. Regularly using the home test device will thusenable the subject to notice a sudden change in levels of said one ormore markers as compared to the base-line level, which can enable him tocontact a medical practitioner.

Alternatively, the reference value can be determined in the subjectsuffering from a given disease or condition as taught herein, which thenindicates his personal “risk level” for any one or more markers, i.e.the level of said one or more markers which indicates he is or will soonbe exposed to said disease or condition. This risk level is interestingfor monitoring the disease progression or for evaluating the effect ofthe treatment.

Furthermore, the reference value or level can be established throughcombined measurement results in subjects with highly similar diseasestates or phenotypes (e.g. all having no disease or condition as taughtherein or having said disease or condition).

Non-limiting examples of semi-quantitative tests known in the art, theprinciple of which could be used for the home test device according tothe present invention are the HIV/AIDS test or Prostate Cancer testssold by Sanitoets. The home prostate test is a rapid test intended as aninitial semi-quantitative test to detect PSA blood levels higher than 4ng/ml in whole blood. The typical home self-test kit comprises thefollowing components: a test device to which the blood sample is to beadministered and which results in a signal when the protein level isabove a certain threshold level, an amount of diluent e.g. in dropperpipette to help the transfer of the analytes (i.e. the protein ofinterest) from the sample application zone to the signal detection zone,optionally an empty pipette for blood specimen collection, a fingerpricking device, optionally a sterile swab to clean the area of prickingand instructions of use of the kit.

Similar tests are also known for e.g. breast cancer detection andCRP-protein level detection in view of cardiac risk home tests. Thelatter test encompasses the sending of the test result to a laboratory,where the result is interpreted by a technical or medical expert. Suchtelephone or internet based diagnosis of the patient's condition is ofcourse possible and advisable with most of the kits, sinceinterpretation of the test result is often more important thanconducting the test. When using an electronic device as mentioned abovewhich gives a numerical value of the level of protein present in thesample, this value can of course easily be communicated throughtelephone, mobile telephone, satellite phone, E-mail, internet or othercommunication means, warning a hospital, a medicinal practitioner or afirst aid team that a person is, or may be at risk of, suffering fromthe disease or condition as taught herein. A non-limiting example ofsuch a system is disclosed in U.S. Pat. No. 6,482,156.

The presence and/or concentration of PERLECAN in a sample can bemeasured by surface plasmon resonance (SPR) using a chip having PERLECANbinding molecule immobilized thereon, fluorescence resonance energytransfer (FRET), bioluminescence resonance energy transfer (BRET),fluorescence quenching, fluorescence polarization measurement or othermeans known in the art. Any of the binding assays described can be usedto determine the presence and/or concentration of PERLECAN in a sample.To do so, PERLECAN binding molecule is reacted with a sample, and theconcentration of PERLECAN is measured as appropriate for the bindingassay being used. To validate and calibrate an assay, control reactionsusing different concentrations of standard PERLECAN and/or PERLECANbinding molecule can be performed. Where solid phase assays areemployed, after incubation, a washing step is performed to removeunbound PERLECAN. Bound, PERLECAN is measured as appropriate for thegiven label (e.g., scintillation counting, fluorescence, antibody-dyeetc.). If a qualitative result is desired, controls and differentconcentrations may not be necessary. Of course, the roles of PERLECANand PERLECAN binding molecule may be switched; the skilled person mayadapt the method so PERLECAN binding molecule is applied to sample, atvarious concentrations of sample.

A PERLECAN binding molecule according to the invention is any substancethat binds specifically to PERLECAN. Examples of a PERLECAN bindingmolecule useful according to the present invention, includes, but is notlimited to an antibody, a polypeptide, a peptide, a lipid, acarbohydrate, a nucleic acid, peptide-nucleic acid, small molecule,small organic molecule, or other drug candidate. A PERLECAN bindingmolecule can be natural or synthetic compound, including, for example,synthetic small molecule, compound contained in extracts of animal,plant, bacterial or fungal cells, as well as conditioned medium fromsuch cells. Alternatively, PERLECAN binding molecule can be anengineered protein having binding sites for PERLECAN. According to anaspect of the invention, a PERLECAN binding molecule binds specificallyto PERLECAN with an affinity better than 10⁻⁶ M. A suitable PERLECANbinding molecule e can be determined from its binding with a standardsample of PERLECAN. Methods for determining the binding between PERLECANbinding molecule and PERLECAN are known in the art. As used herein, theterm antibody includes, but is not limited to, polyclonal antibodies,monoclonal antibodies, humanised or chimeric antibodies, engineeredantibodies, and biologically functional antibody fragments (e.g. scFv,nanobodies, Fv, etc) sufficient for binding of the antibody fragment tothe protein. Such antibody may be commercially available antibodyagainst PERLECAN, such as, for example, a mouse, rat, human or humanisedmonoclonal antibody.

In a preferred embodiment, the binding molecule or agent is capable ofbinding both the mature membrane- or cell-bound PERLECAN protein orfragment. In a more preferred embodiment, the binding agent or moleculeis specifically binding or detecting the soluble form, preferably theplasma circulating form of PERLECAN, as defined herein.

According to one aspect of the invention, the PERLECAN binding moleculeis labelled with a tag that permits detection with another agent (e.g.with a probe binding partner). Such tags can be, for example, biotin,streptavidin, his-tag, myc tag, maltose, maltose binding protein or anyother kind of tag known in the art that has a binding partner. Exampleof associations which can be utilised in the probe:binding partnerarrangement may be any, and includes, for example biotin:streptavidin,his-tag:metal ion (e.g. Ni²⁺), maltose:maltose binding protein.

The specific-binding agents, peptides, polypeptides, proteins,biomarkers etc. in the present kits may be in various forms, e.g.,lyophilised, free in solution or immobilised on a solid phase. They maybe, e.g., provided in a multi-well plate or as an array or microarray,or they may be packaged separately and/or individually. The may besuitably labelled as taught herein. Said kits may be particularlysuitable for performing the assay methods of the invention, such as,e.g., immunoassays, ELISA assays, mass spectrometry assays, and thelike.

The term “modulate” generally denotes a qualitative or quantitativealteration, change or variation specifically encompassing both increase(e.g., activation) or decrease (e.g., inhibition), of that which isbeing modulated. The term encompasses any extent of such modulation.

For example, where modulation effects a determinable or measurablevariable, then modulation may encompass an increase in the value of saidvariable by at least about 10%, e.g., by at least about 20%, preferablyby at least about 30%, e.g., by at least about 40%, more preferably byat least about 50%, e.g., by at least about 75%, even more preferably byat least about 100%, e.g., by at least about 150%, 200%, 250%, 300%,400% or by at least about 500%, compared to a reference situationwithout said modulation; or modulation may encompass a decrease orreduction in the value of said variable by at least about 10%, e.g., byat least about 20%, by at least about 30%, e.g., by at least about 40%,by at least about 50%, e.g., by at least about 60%, by at least about70%, e.g., by at least about 80%, by at least about 90%, e.g., by atleast about 95%, such as by at least about 96%, 97%, 98%, 99% or even by100%, compared to a reference situation without said modulation.

Preferably, modulation of the activity and/or level of intendedtarget(s) (e.g., PERLECAN gene or protein) may be specific or selective,i.e., the activity and/or level of intended target(s) may be modulatedwithout substantially altering the activity and/or level of random,unrelated (unintended, undesired) targets.

Reference to the “activity” of a target such as PERLECAN protein maygenerally encompass any one or more aspects of the biological activityof the target, such as without limitation any one or more aspects of itsbiochemical activity, enzymatic activity, signalling activity and/orstructural activity, e.g., within a cell, tissue, organ or an organism.

In the context of therapeutic or prophylactic targeting of a target, thereference to the “level” of a target such PERLECAN gene or protein maypreferably encompass the quantity and/or the availability (e.g.,availability for performing its biological activity) of the target,e.g., within a cell, tissue, organ or an organism.

For example, the level of a target may be modulated by modulating thetarget's expression and/or modulating the expressed target. Modulationof the target's expression may be achieved or observed, e.g., at thelevel of heterogeneous nuclear RNA (hnRNA), precursor mRNA (pre-mRNA),mRNA or cDNA encoding the target. By means of example and notlimitation, decreasing the expression of a target may be achieved bymethods known in the art, such as, e.g., by transfecting (e.g., byelectroporation, lipofection, etc.) or transducing (e.g., using a viralvector) a cell, tissue, organ or organism with an antisense agent, suchas, e.g., antisense DNA or RNA oligonucleotide, a construct encoding theantisense agent, or an RNA interference agent, such as siRNA or shRNA,or a ribozyme or vectors encoding such, etc. By means of example and notlimitation, increasing the expression of a target may be achieved bymethods known in the art, such as, e.g., by transfecting (e.g., byelectroporation, lipofection, etc.) or transducing (e.g., using a viralvector) a cell, tissue, organ or organism with a recombinant nucleicacid which encodes said target under the control of regulatory sequenceseffecting suitable expression level in said cell, tissue, organ ororganism. By means of example and not limitation, the level of thetarget may be modulated via alteration of the formation of the target(such as, e.g., folding, or interactions leading to formation of acomplex), and/or the stability (e.g., the propensity of complexconstituents to associate to a complex or disassociate from a complex),degradation or cellular localisation, etc. of the target.

In a preferred embodiment, said modulation leads to a decrease inPERLECAN activity, either by inactivating or blocking its function it atthe protein level or by preventing transcription and translation of thecoding sequence of PERLECAN into its protein, i.e. at the mRNA or genelevel. Since it is clear that the PERLECAN level is increased insubjects suffering form renal dysfunction as defined herein, decreasingthe activity of PERLECAN intends to normalise and/o improve thecondition of the subject.

The term “antisense” generally refers to a molecule designed tointerfere with gene expression and capable of specifically binding to anintended target nucleic acid sequence. Antisense agents typicallyencompass an oligonucleotide or oligonucleotide analogue capable ofspecifically hybridising to the target sequence, and may typicallycomprise, consist essentially of or consist of a nucleic acid sequencethat is complementary or substantially complementary to a sequencewithin genomic DNA, hnRNA, mRNA or cDNA, preferably mRNA or cDNAcorresponding to the target nucleic acid. Antisense agents suitableherein may typically be capable of hybridising to their respectivetarget at high stringency conditions, and may hybridise specifically tothe target under physiological conditions.

The term “ribozyme” generally refers to a nucleic acid molecule,preferably an oligonucleotide or oligonucleotide analogue, capable ofcatalytically cleaving a polynucleotide. Preferably, a “ribozyme” may becapable of cleaving mRNA of a given target protein, thereby reducingtranslation thereof. Exemplary ribozymes contemplated herein include,without limitation, hammer head type ribozymes, ribozymes of the hairpintype, delta type ribozymes, etc. For teaching on ribozymes and designthereof, see, e.g., U.S. Pat. No. 5,354,855, U.S. Pat. No. 5,591,610,Pierce et al. 1998 (Nucleic Acids Res 26: 5093-5101), Lieber et al. 1995(Mol Cell Biol 15: 540-551), and Benseler et al. 1993 (J Am Chem Soc115: 8483-8484).

“RNA interference” or “RNAi” technology is routine in the art, andsuitable RNAi agents intended herein may include inter alia shortinterfering nucleic acids (siNA), short interfering RNA (sRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules as known in the art. For teaching on RNAi moleculesand design thereof, see inter alia Elbashir et al. 2001 (Nature 411:494-501), Reynolds et al. 2004 (Nat Biotechnol 22: 326-30), Wang & Mu2004 (Bioinformatics 20: 1818-20), Yuan et al. 2004 (Nucleic Acids Res32(Web Server issue): W130-4), by M Sohail 2004 (“Gene Silencing by RNAInterference: Technology and Application”, 1^(st) ed., CRC, ISBN0849321417), U Schepers 2005 (“RNA Interference in Practice: Principles,Basics, and Methods for Gene Silencing in C. elegans, Drosophila, andMammals”, 1^(st) ed., Wiley-VCH, ISBN 3527310207), and DR Engelke & J JRossi 2005 (“Methods in Enzymology, Volume 392: RNA Interference”,1^(st) ed., Academic Press, ISBN 0121827976).

The term “pharmaceutically acceptable” as used herein is consistent withthe art and means compatible with the other ingredients of apharmaceutical composition and not deleterious to the recipient thereof.

As used herein, “carrier” or “excipient” includes any and all solvents,diluents, buffers (such as, e.g., neutral buffered saline or phosphatebuffered saline), solubilisers, colloids, dispersion media, vehicles,fillers, chelating agents (such as, e.g., EDTA or glutathione), aminoacids (such as, e.g., glycine), proteins, disintegrants, binders,lubricants, wetting agents, emulsifiers, sweeteners, colorants,flavourings, aromatisers, thickeners, agents for achieving a depoteffect, coatings, antifungal agents, preservatives, antioxidants,tonicity controlling agents, absorption delaying agents, and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active substance, its use in the therapeuticcompositions may be contemplated.

The present active substances (agents) may be used alone or incombination with any therapies known in the art for the disease andconditions as taught herein (“combination therapy”). Combinationtherapies as contemplated herein may comprise the administration of atleast one active substance of the present invention and at least oneother pharmaceutically or biologically active ingredient. Said presentactive substance(s) and said pharmaceutically or biologically activeingredient(s) may be administered in either the same or differentpharmaceutical formulation(s), simultaneously or sequentially in anyorder.

The dosage or amount of the present active substances (agents) used,optionally in combination with one or more other active compound to beadministered, depends on the individual case and is, as is customary, tobe adapted to the individual circumstances to achieve an optimum effect.Thus, it depends on the nature and the severity of the disorder to betreated, and also on the sex, age, body weight, general health, diet,mode and time of administration, and individual responsiveness of thehuman or animal to be treated, on the route of administration, efficacy,metabolic stability and duration of action of the compounds used, onwhether the therapy is acute or chronic or prophylactic, or on whetherother active compounds are administered in addition to the agent(s) ofthe invention.

Without limitation, depending on the type and severity of the disease, atypical daily dosage might range from about 1 μg/kg to 100 mg/kg of bodyweight or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. A preferred dosage of the active substance of theinvention may be in the range from about 0.05 mg/kg to about 10 mg/kg ofbody weight. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0mg/kg or 10 mg/kg (or any combination thereof) may be administered tothe patient. Such doses may be administered intermittently, e.g., everyweek or every two or three weeks.

As used herein, a phrase such as “a subject in need of treatment”includes subjects that would benefit from treatment of a given diseaseor condition as taught herein. Such subjects may include, withoutlimitation, those that have been diagnosed with said condition, thoseprone to contract or develop said condition and/or those in whom saidcondition is to be prevented.

The terms “treat” or “treatment” encompass both the therapeutictreatment of an already developed disease or condition, as well asprophylactic or preventative measures, wherein the aim is to prevent orlessen the chances of incidence of an undesired affliction, such as toprevent the chances of contraction and progression of a disease orcondition as taught herein. Beneficial or desired clinical results mayinclude, without limitation, alleviation of one or more symptoms or oneor more biological markers, diminishment of extent of disease,stabilised (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and the like. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

The term “prophylactically effective amount” refers to an amount of anactive compound or pharmaceutical agent that inhibits or delays in asubject the onset of a disorder as being sought by a researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” as used herein, refers to an amountof active compound or pharmaceutical agent that elicits the biologicalor medicinal response in a subject that is being sought by a researcher,veterinarian, medical doctor or other clinician, which may include interalia alleviation of the symptoms of the disease or condition beingtreated. Methods are known in the art for determining therapeuticallyand prophylactically effective doses for the present compounds.

The above aspects and embodiments are further supported by the followingnon-limiting examples.

EXAMPLES Example 1 MASSTERCLASS Targeted Protein Quantitation for EarlyValidation of Candidate Markers Derived from Discovery

MASSTERCLASS Experimental Setup

MASSTERCLASS assays use targeted tandem mass spectrometry with stableisotope dilution as an end-stage peptide quantitation system (alsocalled Multiple Reaction Monitoring (MRM) and Single Reaction Monitoring(SRM). The targeted peptide is specific (i.e., proteotypic) for thespecific protein of interest. i.e., the amount of peptide measured isdirectly related to the amount of protein in the original sample. Toreach the specificity and sensitivity needed for biomarker quantitationin complex samples, peptide fractionations precede the end-stagequantitation step.

A suitable MASSTERCLASS assay may include the following steps:

-   -   Plasma/serum sample    -   Depletion of human albumin and IgG (complexity reduction on        protein level) using affinity capture with anti-albumin and        anti-IgG antibodies using ProteoPrep spin columns (Sigma        Aldrich)    -   Spiking of known amounts of isotopically labelled peptides. This        peptide has the same amino acid sequence as the proteotypic        peptide of interest, typically with one isotopically labelled        amino acid built in to generate a mass difference. During the        entire process, the labelled peptide has identical chemical and        chromatographic behaviour as the endogenous peptide, except        during the end-stage quantitation step which is based on        molecular mass.    -   Tryptic digest. The proteins in the depleted serum/plasma sample        are digested into peptides using trypsin. This enzyme cleaves        proteins C-terminally from lysine and argninine, except when a        proline is present C-terminally of the lysine or arginine.        Before digestion, proteins are denatured by boiling, which        renders the protein molecule more accessible for the trypsin        activity during the 16 h incubation at 37° C.    -   First peptide-based fractionation: Free Flow Electrophoresis        (FFE; BD Diagnostic) is a gel-free, fluid separation technique        in which charged molecules moving in a continuous laminar flow        are separated through an electrical field perpendicular to the        flow. The electrical field causes the charged molecules to        separate in the pH gradient according to their isoelectric point        (pp. Only those fractions containing the monitored peptides are        selected for further fractionation and LC-MS/MS analysis. Each        peptide of interest elutes from the FFE chamber at a specific        fraction number, which is determined during protein assay        development using the synthetic peptide homologue. Specific        fractions or fraction pools (multiplexing) proceed to the next        level of fractionation.    -   Second peptide-based fractionation: Phenyl HPLC (XBridge Phenyl;        Waters) separates peptides according to hydrophobicity and        aromatic nature of amino acids present in the peptide sequence.        Orthogonality with the back-end C18 separation is achieved by        operating the column at an increased pH value (pH 10). As        demonstrated by Gilar et al. 2005, J Sep Sci 28(14): 1694-1703),        pH is by far the most drastic parameter to alter peptide        selectivity in RP-HPLC. Each peptide of interest elutes from the        Phenyl column at a specific retention time, which is determined        during protein assay development using the synthetic peptide        homologue. The use of an external control system, in which a        mixture of 9 standard peptides is separated upfront a batch of        sample separations, allows adjusting the fraction collection in        order to correct for retention time shifts. The extent of        fractionation is dependent on the concentration of the protein        in the sample and the complexity of that sample.    -   LC-MS/MS based quantitation, including further separation on        reversed phase (C18) nanoLC (PepMap C18; Dionex) and MS/MS:        tandem mass spectrometry using MRM (4000 QTRAP; ABI)/SRM        (Vantage TSQ; Thermo Scientific) mode. The LC column is        connected to an electrospray needle connected to the source head        of the mass spectrometer. As material elutes from the column,        molecules are ionized and enter the mass spectrometer in the gas        phase. The peptide that is monitored is specifically selected to        pass the first quadrupole (Q1), based on its mass to charge        ratio (m/z). The selected peptide is then fragmented in a second        quadrupole (Q2) which is used as a collision cell. The resulting        fragments then enter the third quadrupole (Q3). Depending on the        instrument settings (determined during the assay development        phase) only a specific peptide fragment or specific peptide        fragments (or so called transitions) are selected for detection.    -   The combination of the m/z of the monitored peptide and the m/z        of the monitored fragment of this peptide is called a        transition. This process can be performed for multiple        transitions during one experiment. Both the endogenous peptide        (analyte) and its corresponding isotopically labelled synthetic        peptide (internal standard) elute at the same retention time,        and are measured in the same LC-MS/MS experiment.    -   The MASSTERCLASS readout is defined by the ratio between the        area under the peak specific for the analyte and the area under        the peak specific for the synthetic isotopically labelled        analogue (internal standard). MASSTERCLASS readouts are directly        related to the original concentration of the protein in the        sample. MASSTERCLASS readouts can therefore be compared between        different samples and groups of samples.

A typical MASSTERCLASS protocol followed in the present study is givenhere below:

-   -   25 μL of plasma is subjected to a depletion of human albumin and        IgG (ProteoPrep spin columns; Sigma Aldrich) according to the        manufacturer's protocol, except that 20 mM NH₄HCO₃ was used as        the binding/equilibration buffer.    -   The depleted sample (225 μL) is denatured for 15 min at 95° C.        and immediately cooled on ice    -   500 fmol of the isotopically labelled peptide (custom made        ‘Heavy AQUA’ peptide; Thermo Scientific) is spiked in the sample    -   20 μg trypsin is added to the sample and digestion is allowed        for 16 h at 37° C.    -   The digested sample was first diluted 1/8 in solvent A (0.1%        formic acid) and then 1/20 in the same solvent containing 250        amol/μL of all isotopically labelled peptides (custom made        ‘Heavy AQUA’ peptide; Thermo Scientific) of interest.    -   20 μL of the final dilution was separated using reverse-phase        NanoLC with on-line MS/MS in MRM/SRM mode:        -   Column: PepMap C18, 75 μm I.D.×25 cm L, 100 Å pore diameter,            5 μm particle size        -   Solvent A: 0.1% formic acid        -   Solvent B: 80% acetonitrile, 0.1% formic acid        -   Gradient: 30 min; 2%-55% Solvent B        -   MS/MS in MRM mode: method contains the transitions for the            analyte as well as for the synthetic, labelled peptide.        -   The used transitions were experimentally determined and            selected during protein assay development        -   Each of the transitions of interest was measured for a            period starting 3 minutes before and ending 3 minutes after            the determined retention time of the peptide of interest,            making sure that each peak had at least 15 datapoints.    -   The raw data was analysed and quantified using the LCQuan        software (Thermo Scientific): the area under the analyte (=the        PERLECAN peptide) peak and under the internal standard (the        labelled, synthetic PERLECAN peptide) peak at the same C18        retention time was determined by automatic peak detection. These        were cross-checked manually.    -   The MASSTERCLASS readout was defined by the ratio of the analyte        peak area and the internal standard peak area

MASSTERCLASS Output

The measured ratios are differential quantitations of peptides. In otherwords a ratio is the normalised concentration of a peptide. Theconcentration of a peptide is proportional to the ratio measured in themass spectrometer.

Example 2 Screening of Acute Dyspnea Samples for PERLECAN

In this example the clinical utility of PERLECAN measurement for theevaluation of dyspneic patients was assessed.

The 299 clinical samples used in this study are part of the BASEL Vcohort, a prospective study on consecutive patients presentingthemselves to the ED of the university Hospital of BASEL with dyspnea asthe most prominent symptom (part of this cohort is described in Potockiet al., Journal of Internal Medicine 2010 January; 267(1):119-29). Thegold standard for the diagnosis of acute heart failure was basedinterpretation of two independent cardiologists of all medical recordspertaining to the patient including 90 day follow up data and BNPlevels. Based on this, 56% (n=168) of patients were adjudicated to havean acute heart failure event, others were classified as dyspneanon-heart failure. A wide range of clinical and marker variables wascollected (for summary see Table 1) including patient demographics,medical history, chronic medication, renal function parameters, echoparameters, established cardiac and inflammatory marker levels.Glomerular filtration rate was calculated using the Modification of Dietin Renal Disease (MDRD) formula (Stevens et al., New England Journal ofMedicine 2006; 354:2473-83). Patients were followed up for at least 1year post admission to the hospital and all-cause-mortality wasrecorded.

PERLECAN and Cystatin C levels were measured using MASSTERCLASS™ assaysas described in example 1. BNP, NT-proBNP and CRP levels were measuredusing commercially available immunoassays as described in Potocki et al(2010).

The diagnostic accuracy of a specific protein was determined bymeasuring the area under the Receiver-Operating-Characteristics (ROC)curves (AUC) as in Sullivan Pepe M (The statistical evaluation ofmedical tests for classification and prediction. 1993 Oxford UniversityPress New York). The estimated and confidence intervals for AUCs werealso computed using a nonparametric approach, namely bootstrapping(Efron B, Tibshirani R J. Nonparametric confidence intervals. Anintroduction to the bootstrap. Monographs on statistics and appliedprobability. 1993; 57:75-90 Chapman & Hall New York).

Associations of PERLECAN, Cystatin C, BNP, NT-proBNP and CRP levels withall available clinical parameters were computed using univariatestatistical tests. Spearman's ranked test was used to computecorrelation coefficients and Wilcoxon rank sum test for assessingwhether two independent samples of observation originate from the samepopulation.

TABLE 1 Summary of patient characteristics included in the study. allpatients Characteristic (n = 299) age (yr) 77 gender (% male) 52 BMI 26History (%) hypertension 68 heart failure 24 CAD 28 diabetes 18 COPD 34chronic kidney 28 disease physical/ECG heart rate 93 ± 23 systolic bp138 ± 26  diastolic bp 83 ± 16 LVEF 24 (20-28)  lab s creatinin (umol/L)85 (66-120) eGFR 67 (44-89)  (mL/min/1.73 m2) BNP (pg/mL) 350 (90-1120)Nt-proBNP (pg/mL) 1656 (314-6105) diagnosis (%) ADHF 56% Pneumonia 10%Pulmonary  3% embolism COPD/Asthma 16% hyperventilation  3% other 12%outcome survival at 1 yr 73%

Example 3 PERLECAN Associates with Kidney Function Parameters

Screening acute dyspnea patients (example 2) for PERLECAN levels showeda clear association of PERLECAN level with all available clinicalparameters related to kidney function as indicated by the low p-valuesfor Spearman rank correlation with estimated glomerular filtration rate(eGfr), creatinine levels and blood urea nitrogen (BUN) levels and thelow Wilcoxon p-values for presence/absence of history of kidney failure(summarized in Table 2). FIG. 2 illustrates the correlation of PERLECANwith eGfr, indicating PERLECAN is a good indicator of glomerularfiltration. The correlation of PERLECAN with filtration is furthercorroborated by correlation with Cystatin C, a known good marker forGfr. Cystatin C was also measured in these samples using MASSTERCLASStechnology. The correlation of PERLECAN with Cystatin C and eGfr remainsvalid after correcting for presence of acute decompensated heart failure(Table 2).

Table 2: Summary statistics on univariate associations for Cystatin Cand PERLECAN. Values mentioned for Spearman ranked test are correlationcoefficients between 2 continuous variables, Wilcoxon test returnsp-values which show significance of association between continuousmarker levels and 2 discrete populations.

Cohort Population1 Population 2 PERLECAN BASEL AHF (n = 85) Dyspnea nonAHF (n = 69) 0.76 (0.68-0.83) BASEL Kidney No kidney failure (n = 115)0.91 (0.83-0.97) failure (n = 40) population PERLECANhistory_kidney.failure AHF 1.16E−07 history_kidney.failure dyspnea nonAHF 0.004415 lab_creatinine AHF 4.78E−12 lab_creatinine dyspnea non AHF3.94E−08 lab_creatinine.bin.150 AHF 3.77E−08 lab_creatinine.bin.150dyspnea non AHF 0.005128 lab_creatinine.bin.250 AHFlab_creatinine.bin.250 dyspnea non AHF lab_gfr AHF 1.34E−11 lab_gfrdyspnea non AHF 3.70E−08 lab_urea.bin.7.5 AHF 2.16E−08 lab_urea.bin.7.5dyspnea non AHF 0.000257 Cystatin C_MC089_B AHF 0 Cystatin C_MC089_Bdyspnea non AHF 1.27E−08 medication.on.admission_diuretic AHF 5.04E−06medication.on.admission_diuretic dyspnea non AHF

Example 4 PERLECAN as a Marker of Renal Dysfunction

Estimated glomerular filtration rate is a good indicator of how well thekidneys are functioning. Patients with eGfr below 60 for a minimum of 3months are considered to have chronic kidney disease (CKD). The acutedyspnea population under study has 107 patients with reduced eGfr vs 192patients with more normal glomerular filtration rates.

Receiver-operating characteristics (ROC) analysis demonstrated PERLECANto be highly sensitive and specific for diagnosing kidney dysfunction inthis population of dyspneic patients, as indicated by an overall medianAUC of 0.9 with 95% CI 0.85-0.93 (FIG. 3). This diagnostic performanceis equivalent to Cystatin C, the best available biomarker for chronickidney disease.

FIG. 4 illustrates relative levels of PERLECAN as measured byMASSTERCLASS in patients with reduced (<60), intermediate (60-90) andnormal (>90) estimated glomerular filtration rates. Median PERLECANlevels among patients with patients with reduced eGfr were 4.7 foldhigher than patients with normal eGfr (>90). PERLECAN levels are alsoelevated in patients with slightly reduced kidney function (eGfr between60 and 90).

Example 5 PERLECAN as a Marker for Acute Changes in Renal Function

The correlation of PERLECAN with Cystatin C and eGfr remains valid aftercorrecting for presence of acute decompensated heart failure. Thecorrelation in AHF patients hints to the fact that PERLECAN can detectacute changes in eGfr due to the acute decompensation of the heart(i.e., reduced cardiac output).

After restoring cardiac output and renal function by therapeuticintervention PERLECAN levels also return to baseline levels.

Example 6 PERLECAN as Predictive Marker for Mortality

In the cohort of acute dyspnea patients under study patients werefollowed up for at least one year post admission. At 1 year postadmission, 82/299 subjects (27%) had died (all-cause mortality). Therelation of PERLECAN and other clinical and marker variables tomortality was studied using different methods. Receiver-operatorcharacteristic analysis with death at 1 year as the reference standardwere performed and median area under the curve was calculated.Distributions of marker levels in “alive” and “death” patients werecompared using the Wilcoxon rank-sum test. Kaplan Meier curves comparedmortality rates across the follow-up period after presentation in groupsdivided as a function of PERLECAN levels.

Concentrations of PERLECAN at presentation in patients with acutedyspnea were significantly higher among patients who died by 1 year(n=82; 27%) compared with patients who were alive (p=2e⁻¹¹). Thispattern of higher PERLECAN concentrations in decedents remained whensubjects were considered as a function of the presence (p=3.5e⁻⁰⁸) orabsence of acute heart failure (p=0.01) and when the population wasdivided based on renal function (eGfr<60; p=8.8e-05 vs eGfr>60;p=0.0003). This illustrates PERLECAN has the potential to predict badoutcome in a general dyspneic population as well as in an acute heartfailure population and a chronic kidney disease population.

In addition decile analysis of PERLECAN concentrations examined as afunction of mortality rates at 1 year revealed that there was a gradedincrease in mortality with rising concentrations of the marker. ROCanalysis performed for predicting death at 1 year in all acute dyspneapatients demonstrated an AUC of 0.74 for PERLECAN (95% CI: 0.64-0.83),similar to NT-proBNP (AUC=0.77, but higher than BNP, Cystatin C and CRPprotein markers (FIG. 5).

Example 7 PERLECAN Predicts Renal Replacement Therapy Requirement inPostoperative Patients

PERLECAN levels were further studied in a cohort of cardiac surgerypatients (n=100), both pre and 24 h post surgery. Patients were selectedto include 50 patients who did develop AKI during follow-up matched with50 controls. Of the 100 patients, 12% required renal replacement therapy(RRT) during follow-up. Table 3 summarizes the patient characteristicsincluded in this study. The peptides detected are represented by SEQ IDNos 4 and 6.

TABLE 3 Overview of patient characteristics that underwent cardiacsurgery Variables No RRT (n = 88) RRT (n = 12) P-value Age (yrs) 69.5 73.5 p = 0.04 Age >70 yrs 53% (n = 47) 69% (n = 8) p = 0.22 Gender (%male) 76% (n = 67) 69% (n = 8) p = 0.72 Type of surgery CABG 55% (n =48) 33% (n = 4) p = 0.2 Valve repair/replacement 72% (n = 63) 67% (n =8) p = 0.7 concomitant 30% (n = 26) 25% (n = 3) p = 1 Pre-op kidneyfunction eGfr 63.8 (54-77) 47.5 (41-67) p = 0.07 Serum creatinin 88.4122.4 p = 0.06 Serum creatinin >120 50% (n = 6) umol/L Medical historyNYHA III or IV 26% (n = 23)  0 p = 1 LVEF 52.5 (39.25-60)   50 (45-60) p= 0.97 COPD 17% (n = 15) 33% (n = 4) P = 0.25 IDDM  6% (n = 5)  0 P =0.61 Outcome AKI 44% 91% (n = 10) 0.004

PERLECAN, Cystatin C, lactate, NGAL (plasma and urine) and serumcreatinine levels were measured pre-surgery (at induction) and 24 hrspost surgery. PERLECAN and Cystatin C were measured using MASSTERCLASS,urine NGAL was measured on the ABBOTT Architect (ABBOTT diagnostics,Germany), plasma NGAL was measured using the Bioporto NGAL assay(Bioporto diagnostics, Denmark). The performances of the differentmarkers to predict RRT requirement was calculated and are summarized intable 4. FIG. 6 illustrates the performance of PERLECAN compared tocreatinine based prediction at different timepoints pre and postsurgery. PERLECAN measured at 24 h post surgery is a good predictor ofRRT requirement and is at this timepoint 3-4 days earlier to do so thancreatinine based predictions. At the 24 h timepoint PERLECAN hassignificantly better performance than creatinine, or eGfr. Alsopre-surgery already PERLECAN shows a better performance to predict RRTrequirement.

TABLE 4 Performance of the different markers to predict RRT requirementboth before and 24 h after surgery. timepoint protein/marker AUC (95%CI) before Ngal (plasma) 0.69 (0.50-0.84) before Ngal (urine) 0.52(0.30-0.74) before lactate 0.58 (0.43-0.72) before Cystatin C 0.76(0.61-0.88) before PERLECAN 0.77 (0.62-0.88) 24 h_post Ngal (plasma)0.73 (0.55-0.88) 24 h_post Ngal (urine) 0.58 (0.38-0.78) 24 h_postlactate 0.68 (0.48-0.85) 24 h_post creatinin 0.75 (0.59-0.88) 24 h_postCystatin C 0.87 (0.78-0.94) 24 h_post PERLECAN 0.89 (0.81-0.95)

Example 8 Different PERLECAN Peptides May Show Different Relation toKidney Filtration Function

Different MASSTERCLASS assays were built that will quantify differentdomains/chains of the PERLECAN molecule (see table 5 and FIG. 7). Theassay based on Seq ID no 7 is specific for the large PERLECAN moleculewhile the assays based on Seq IDs 4 and 6 will target the LG3 peptide, areported biologically active fragment of PERLECAN. The assays targetedtowards LG3 peptide are however not specific for this peptide and willalso quantify endorepellin and the entire PERLECAN molecule as well.

The different assays were used to measure kidney filtration function ina cohort of type I diabetes patients. These patients have a good spreadof eGfr, ranging from normal to severely impaired kidney function witheGfr below 40 ml/min/1.72 m2. The levels as measured with the LG3 assaycorrelate again nicely with eGfr (see FIG. 7) and this is true for bothLG3 assays. In this particular experiment, the PERLECAN specific assayshows comparatively poorer correlation with eGfr and other filtrationfunction related markers such as Cystatin C and beta-trace. Thereforethe observed filtration function may probably stem from the LG3 peptide(and/or endorepellin molecule) and not from the entire PERLECANmolecule.

TABLE 5 SEQ Sequence Location Chain ID NO GSIQVDGEELVSGR 4305-4318LG3 domain 4 SLPEVPETIELEVR 4222-4235 LG3 domain 6 LEGDTLIIPR 3258-3267Perlecan domain 7

The results are depicted in FIG. 7 and in table 6 below.

TABLE 6 Correlation coefficient LG3/endorepellin r² Perlecan assay assayCystatin C 0.216 0.923 LG3/endorepellin 0.214 0.937 Beta trace 0.1620.897

The invention claimed is:
 1. A method for treating dyspnea in a subjectpresenting with rapid onset of the dyspnea, wherein the subject has nothad a kidney transplant, and wherein the method comprises: a)identifying the subject presenting with rapid onset of said dyspnea ashaving dyspnea associated with or caused by renal dysfunction and asbeing in need of treatment of renal dysfunction by having an assayconducted, wherein the assay comprises: (i) contacting an antibody or anaptamer capable of specifically detecting the Endorepellin domain ofPERLECAN with a blood sample from the subject; (ii) measuring a quantityof the Endorepellin domain of PERLECAN in the blood sample bydetermining the binding of the antibody or aptamer to the Endorepellindomain of PERLECAN; (iii) converting the measured quantity of theEndorepellin domain of PERLECAN in the blood sample to a glomerularfiltration rate (GFR) value; and (iv) comparing the GFR value determinedin (iii) with a reference value GFR value, said reference valuerepresenting one or more of a known diagnosis, prediction, or prognosisof renal dysfunction or normal renal function, and finding a reduced GFRvalue determined in (iii) from said reference value so as to identifythe subject as being in need of the treatment for renal dysfunction; andb) treating the subject having the deviation for renal dysfunction,thereby treating dyspnea, with a treatment or therapy selected from thegroup consisting of low-potassium diet, low phosphorous diet, lowpotassium and low phosphorus diet, phosphorous-lowering medications,red-blood-cell-production-stimulating agents, iron supplements, bloodpressure medications, vitamin supplements, haemodialysis,ultrafiltration, peritoneal dialysis and kidney transplantation.
 2. Themethod according to claim 1, wherein dyspnea is associated or caused byrenal dysfunction, wherein the renal dysfunction is acute renal failure,chronic renal failure, is associated or caused by renal fibrosis, or isassociated with or caused by type II diabetes.
 3. The method accordingto claim 1, wherein the subject is a postoperative patient, a criticallyill patient, or a postoperative and critically ill patient.
 4. Themethod according to claim 1, wherein a threshold between normal andreduced GFR is set at a value between about 50 and about 70 ml/min/1.73m², wherein a value above said threshold denotes normal GFR and a valuebelow said threshold denotes reduced GFR.
 5. The method according toclaim 4, wherein the threshold is set at a value at 60 ml/min/1.73 m².6. The method according to claim 1, wherein the identification step a)further comprises: (i) measuring the quantity of the Endorepellin domainof PERLECAN and one or more of the presence or absence; or quantity ofone or more other biomarkers in the blood sample from the subject; (ii)using the measurements of (i) to establish a subject profile of thequantity of the Endorepellin domain of PERLECAN and one or more of thepresence or absence, or quantity of said one or more other biomarkers inthe subject; (iii) comparing said subject profile of (ii) to a referenceprofile of the quantity of the Endorepellin domain of PERLECAN and oneor more of the presence or absence, or quantity of said one or moreother biomarkers, said reference profile representing one or more of aknown diagnosis, prediction, or prognosis of renal dysfunction or normalrenal function; (iv) finding a deviation or no deviation of the subjectprofile of (ii) from the reference profile; and (v) attributing saidfinding of deviation or no deviation to one or more of a particulardiagnosis, prediction, or prognosis of renal dysfunction in the subjectpresenting with rapid onset of the dyspnea wherein the subject has nothad a kidney transplant.
 7. The method according to claim 6, whereinsaid other biomarker is selected from the group consisting ofcreatinine, Cystatin C, neutrophil gelatinase associated lipocalin(NGAL), beta-trace protein, kidney injury molecule 1 (KIM-1),interleukin-18 (IL-18), B-type natriuretic peptide (BNP), pro-B-typenatriuretic peptide (proBNP), amino terminal pro-B-type natriureticpeptide (NTproBNP), latent transforming growth factor beta bindingprotein 2 (LTBP2) and C-reactive peptide.
 8. The method according toclaim 6, wherein the one or more of quantity, or presence or absence ofthe one or more other biomarkers is measured using a binding agentcapable of specifically binding to said one or more other biomarkers. 9.The method according to claim 6, wherein the one or more of quantity of,or presence or absence of the one or more other biomarkers is measuredusing an immunoassay technology, a mass spectrometry analysis method, achromatography method, or a combination of said methods, or using RNAanalysis tools, wherein the RNA analysis tools are northern blotting,RT-PCR or quantitative RT-PCR.